Compounds that inhibit (block) bitter taste in composition and use thereof

ABSTRACT

The present invention relates to the discovery that specific human taste receptors in the T2R taste receptor family respond to particular bitter compounds present in, e.g., coffee. Also, the invention relates to the discovery of specific compounds and compositions containing that function as bitter taste blockers and the use thereof as bitter taste blockers or flavor modulators in, e.g., coffee and coffee flavored foods, beverages and medicaments. Also, the present invention relates to the discovery of a compound that antagonizes numerous different human T2Rs and the use thereof in assays and as a bitter taste blocker in compositions for ingestion by humans and animals.

RELATED APPLICATIONS

This application is a continuation-in-part of U.S. application Ser. No.12/222,918, filed Aug. 19, 2008, which claims the benefit of U.S.Provisional Application Ser. No. 60/957,129 filed on Aug. 21, 2007 andU.S. Provisional Application Ser. No. 61/047,187, filed on Apr. 23, 2008and relates to U.S. application Ser. No. 11/766,974, which is acontinuation-in-part U.S. application Ser. No. 11/555,617 filed on Nov.1, 2006, which is in turn a continuation-in-part of U.S. applicationSer. No. 10/191,058 filed Jul. 10, 2002, now U.S. Pat. No. 7,338,771 andis also a continuation-in-part of U.S. application Ser. No. 10/742,209filed on Dec. 1, 2003, which is a divisional of U.S. application Ser.No. 09/825,882 filed on Apr. 5, 2001, now U.S. Pat. No. 7,105,650, allof which applications are incorporated by reference in their entiretiesherein.

FIELD OF THE INVENTION

This application relates to the identification of human type 2 tastereceptors (hT2Rs) and the use thereof in assays for the identificationof ligands that activate specific T2Rs. These ligands are useful formodulating taste perception, particularly bitter taste. In our previouspatent applications, we described functional expression of human bittertaste receptors, including hT2R8 and hT2R14. In this patent application,we report that hT2R8 and hT2R14 are activated by a bitter-enrichedfraction of coffee, we also report the identification of antagonists forhT2R8 and hT2R14 using a high throughput screening assay, and thatcombinations of the antagonists can reduce the bitter taste of coffeeand coffee fractions. This invention provides a method to modify andimprove the taste of coffee drinks.

Specifically, the present invention relates to the use of hT2R8 and/orhT2T14 in screening assays and taste tests to identify compounds thatinhibit (block) the bitter taste of coffee and other foods andbeverages.

Also, this invention relates to the discovery of a ligand that has broadbitter antagonistic properties, i.e., it appreciably blocks or inhibitsthe activation of many (13) different bitter receptors by a diverse setof bitter ligands and blocks or inhibits the activation of six otherbitter taste receptors as well as inhibiting bitterness elicited by somebitter compounds for which the bitter receptor(s) that they interactwith has not as yet been elucidated.

More specifically, this invention relates to the discovery of a ligandreferred to herein as compound C that has broad bitter antagonisticproperties, i.e., it appreciably blocks or inhibits the activation ofhT2R3, 7, 10, 14, 16, 44, 51, 55, 61, 63, 64, 65 and 71 bitter tastereceptors by a diverse set of bitter ligands and blocks or inhibits theactivation of six other bitter taste receptors, i.e., hT2R5, 9, 13, 54,67 and 75 as well as inhibiting bitterness elicited by some bittercompounds for which the bitter receptor(s) that they interact with hasnot as yet been elucidated.

Also more specifically the invention provides the discovery that thisantagonist compound reduces the bitter taste of salicin, an hT2R16antagonist, and phenylthiourea a hT2R51 agonist.

Also more specifically this invention provides the discovery that thissame antagonist compound blocks the bitter taste elicited by bittercompounds that activate multiple bitter taste receptors, includingomeprazole that activates hT2R10, 14 and 75; Rebaudioside A, a naturalsweetener that activates at least 7 bitter taste receptors; and thatthis same antagonist further also inhibits bitter taste elicited bybitter compounds wherein the bitter receptor(s) with which they interactis unknown including dextromethorphan and diphenhydramine.

Based thereon, the invention relates to the use of this compound infoods, beverages, medicaments and other ingestibles in order toalleviate the bitter taste thereof, including bitter taste elicited byunidentified bitter ligands or compounds wherein bitterness involves theactivation of multiple bitter receptors or for bitter compounds whereinthe receptor specificity thereof is undetermined.

Also, based thereon the invention relates to the use of this antagonistin order to elucidate a conserved motif present in different human T2Rsthat is involved in ligand binding and T2R activation and the design ofchimeric and mutated G protein-coupled receptors (GPCRs) which areengineered to contain this motif.

Further, the invention relates to any of the compounds identified usingthese screening assays and the use thereof in foods beverages andmedicaments including coffee and coffee flavored foods and beverages andmedicaments.

DESCRIPTION OF THE RELATED ART

One of the basic taste modalities that humans can recognize is bitter.The physiology of bitter taste until quite recently was very poorlyunderstood. Recent studies have started to shed light on the biology oftaste (Lindemann, Nature (2001)). It is now known that many bittercompounds produce bitter taste by interacting with cell surfacereceptors. These receptors belong to the family of seven transmembranedomain receptors that interact with intracellular G proteins. A novelfamily of GPCRs, termed T2Rs, has been identified in humans and rodents(Adler et al., Cell 100(6):693-702 (2000); Chandrashekar et al., Cell100(6): 703-711 (2000); Mats Montmayeur J P, Buck L B. Nature 404(6778):601-4 (2000)). Several lines of evidence prior to the subject inventionsuggested that T2Rs mediate responses to bitter compounds. First, T2Rgenes are specifically expressed in subset of taste receptor cells ofthe tongue and palate epithelia. Second, the gene for one of the humanT2Rs (hT2R1) is located in a chromosomal locus that is linked tosensitivity to bitter compound 6-n-propyl-2-thiouracil in humans (Adleret al., (Id.) (2000)). Third, one of the mouse T2Rs (mT2R5) is locatedin a chromosomal locus that is linked to sensitivity to bitter compoundcycloheximide in mice. It was also shown that mT2R5 can activategustducin, G protein specifically expressed in taste cells and linked tobitter stimuli transduction (Wong et al., Nature 381:796-800 (1996)).Gustducin activation by mT2R5 occurs only in response to cycloheximide(Chandrashekar et al., (Id.) (2000). Thus, it has been proposed thatmT2R family mediates bitter taste response in mice, whereas hT2R familymediates bitter taste response in humans. Only one human T2R wassuggested as having identified bitter ligand-hT2R4 was shown as beingactivated by denatonium (Chandrashekar et al., (Id.) 2000). However,effective denatonium concentrations used in the study (1.5 mM) wereunusually high, i.e., were 10⁵-fold higher than the reported bitterthreshold for denatonium to humans (Saroli, Naturwissenschaften71:428-429 (1984)). Thus, no specific bitter ligand was convincinglymatched to any hT2R. It has been also suggested that each hT2R is ableto bind multiple bitter ligands. This hypothesis is based on the factthat hT2R family consists of only 25 identified members, whereas humanscan recognize hundreds of different compounds as bitter. Sequences ofhT2Rs have been previously reported and are discloses in published PCTapplications by Zuker et al. (WO 01/18050 A2, (2001)) and Adler et al.(WO 01/77676 A1 (2001)) both of which are incorporated by reference intheir entirety herein.

One of the difficulties of studying T2R function is that these receptorsare not readily expressed in cultured mammalian cell lines. To improveT2R expression an N-terminal sequence from well-expressed GPCR,rhodopsin, was attached to T2R sequences (Chandrashekar et al., (Id.)2000). This N-terminal tag also allowed easy monitoring of proteinexpression due to available antibody. In addition, SSTR3 tag (Bufe etal., Nat. Genet. 32:397-400 (2002)), a different N-terminal tag has beenused to improve T2R expression. Whereas the incorporation of therhodopsin tag improved expression of some T2Rs in mammalian cell lines,many of them still were not expressed well enough for functionalstudies. In a different approach mT2R5 was successfully expressed ininsect Sf9 cells and used for functional studies using biochemical GTPγSbinding assay (Chandrashekar et al., (Id.) 2000).

In Applicants' earlier patent application, U.S. application Ser. No.09/825,882 now U.S. Pat. No. 7,105,650, Applicants identified andprovided the nucleic acid sequences and polypeptide sequences for anumber of then-novel human taste receptors including hT2R51, hT2R54,hT2R55, hT2R61, hT2R63, hT2R64, hT2R65, hT2R67, hT2R71, and hT2R75.Additionally in U.S. application Ser. Nos. 11/182,942 and 10/628,464,the entireties of which are incorporated by reference herein, Applicantsprovided the polypeptide and DNA sequence for another identified novelhuman taste receptor named therein hT2R76.

Also, in U.S. application Ser. No. 10/191,058 incorporated by referenceherein in its entirety, Applicants discovered ligands that specificallyactivate three different human T2Rs. Additionally, Applicants recentlyfiled U.S. application Ser. No. 11/455,693, the entirety of which isincorporated by reference herein, which further identified bitterligands that specifically bind to other human T2Rs, and provided relatedassays.

Also, relating to practical utilities of the invention it has beenreported that both T2Rs and T1Rs taste receptors are expressed in thegastrointestinal system. For example, Wu et al., Proc, Natl. Acad. Sci,USA 99(4):2392-7(2002) report that T2Rs are expressed in enterendocrinecells (STC1 cells) as well as gustducin and transducin subunits and thatthese cells likely respond to bitter ligands in the gastrointestinaltract. Also, it has been reported by Chen et al., AM J. Physiol. CellPhysiol. 291(4):C726-39 (2006) that bitter taste stimuli induce Ca++signaling and cholecystokinin (CCK) release in enterendocrine STC-1cells. Also, Rozengurt, A J Physiol Gastrointes Liver Physiol291(2):G171-7 (2006) report that taste receptors in the gut likely playa role in molecular sensing the control of digestive functions, andhormonal and/or neuronal pathways and that they may play a role in thedetection of harmful drugs and survival responses. Further, Sternini AmJ Physiol Gastrointest Liver Physiol. 292(2):G457-61 (2007) report thattaste receptors in the gut may be involved in gastrointestinal functionssuch as molecular sensing, nutrient absorption, protection from harmfulsubstances, and further suggest that an understanding of thesemechanisms may be relevant to disease states and conditions such asfeeding disorders, and inflammation. Further, it has been recentlysuggested by Mace et al., J Physiol. 2007 (Epub) that T2Rs and T1Rsactivate phospholipase C beta 2, PLC beta2, and that there is likely amolecular intestinal sensing system in the gut similar to that presentin lingual cells and that gastrointestinal cells such as brush cells orsolitary chemosensory cells expressing taste receptors may result inGLUT2 increase and may play a role in nutrient sensing, and nutrition inthe treatment of obesity and diabetes. Also, Cui et al, Curr Pharm Des.12(35):4591-600 (2006) suggest that T1Rs expressed in the gut may beused in assays for compounds in treating obesity and diabetes as well asartificial sweeteners.

However, notwithstanding what has been reported and the understandingthat T2R members regulate bitter taste, and their possible role ingastrointestinal functions there exists a need for the identification ofspecific ligands which activate human bitter T2R taste receptors. Agreater understanding of the binding properties of different T2Rs,particularly human T2Rs, would be highly beneficial as it will greaterfacilitate the use thereof in selecting compounds having desired tastemodulatory properties, i.e., which block or inhibit the taste ofspecific bitter compounds. Also, it will provide for the identificationof compounds for treating and modulating gastrointestinal functions andrelated diseases such as obesity, diabetes, food absorption, foodsensing, eating disorders, and in the regulation of related hormones andpeptides such as GLUT2, cholecystokin et al.

SUMMARY OF THE INVENTION

Toward that end, the present invention relates to the discovery thathT2R8 and hT2R14 are activated by a bitter-enriched fraction of coffee.

Also, the present invention relates to the use thereof for theidentification of antagonists for hT2R8 and hT2R14 that inhibit or blockthe bitter taste of coffee and coffee related foods, beverages andmedicaments

Further the invention relates to specific antagonist (bitter blocker)compounds that inhibit the bitter taste of coffee and other coffeeflavored foods, beverages and medicaments.

Also, this invention relates to the discovery of a ligand that has broadbitter antagonistic properties, i.e., it appreciably blocks or inhibitsthe activation of many (13) different bitter receptors by a diverse setof bitter ligands and blocks or inhibits the activation of six otherbitter taste receptors as well as inhibiting bitterness elicited by somebitter compounds for which the bitter receptor(s) that they interactwith has not as yet been elucidated.

More specifically, this invention relates to the discovery of a ligandreferred to herein as compound C that has broad bitter antagonisticproperties, i.e., it appreciably blocks or inhibits the activation ofhT2R3, 7, 10, 14, 16, 44, 51, 55, 61, 63, 64, 65 and 71 bitter tastereceptors by a diverse set of bitter ligands and blocks or inhibits theactivation of six other bitter taste receptors, i.e., hT2R5, 9, 13, 54,67 and 75 as well as inhibiting bitterness elicited by some bittercompounds for which the bitter receptor(s) that they interact with hasnot as yet been elucidated.

Also more specifically the invention provides the discovery that thisantagonist compound reduces the bitter taste of salicin an hT2R16antagonist and phenylthiourea a hT2R51 agonist.

Also more specifically this invention provides the discovery that thissame antagonist compound blocks the bitter taste elicited by bittercompounds that activate multiple bitter taste receptors, includingomeprazole, a compound that activates hT2R10, 14 and 75; Rebaudioside A,a natural sweetener that activates at least 7 bitter taste receptors;and that this same antagonist compound further also inhibits bittertaste elicited by bitter compounds wherein the bitter receptor(s) withwhich they interact is unknown including dextromethorphan anddiphenhydramin.

Based thereon, the invention relates to the use of this and relatedcompounds according to the invention in foods, beverages, medicamentsand other ingestibles in order to alleviate the bitter taste thereof,including bitter tase elicited by unidentified bitter ligands orcompounds wherein bitterness involves the activation of multiple bitterreceptors or bitter compounds wherein the receptor specificity thereofis undetermined.

Also, the present invention relates to foods, beverages and medicamentsthat contain an amount of at least one of the identified bitterantagonist compounds sufficient to inhibit or block the bitter tastethereof.

The inventive discoveries were made using cell-based assays thatmeasured the activity of T2Rs using cells that express a particular T2Rin the presence and absence of specific bitter ligands. In particular,as described in greater detail infra, HEK cell lines expressing theabove-identified specific T2Rs on their surface and which furtherexpressed a chimeric G protein that functionally couple to said T2Rswere used in cell-based assays that detected changes in intracellularcalcium concentrations, and were found to be specifically activated byspecific bitter compounds whereas other hT2Rs were not activated undersimilar conditions.

Therefore, the invention embraces the use of these human taste receptorsin assays, preferably high-throughput assays, to identify othercompounds that modulate, preferably block, the activation of thesereceptors by these and other bitter compounds present in coffee andrelated foods and beverages

Also, the invention relates to the use of these receptors to identifycompounds particularly those present in coffee and coffee flavoredfoods, beverages and medicaments that elicit a bitter taste.

Also, the invention relates to the use of an antagonist compoundpossessing broad ranging antagonist properties for use in vitro assaysand in vivo taste tests to identify bitter compound(s) or bitterfractions for which this compound inhibits the bitter taste elicitedthereby and/or inhibits the activation of one or more bitter tastereceptors by the bitter compound or a fraction containing this bittercompound.

Further the invention specifically relates to the use of this broadacting antagonist in foods, beverages, medicaments and other consumedproducts for ingestion by humans or animals wherein bitter taste isdesirably alleviated.

The invention also embraces assays which include an additional stepwhich evaluates the effect of the identified modulating compounds inhuman or other taste tests, and particularly evaluates the effect of theidentified compounds on bitter taste especially bitter taste elicited bycoffee and fractions derived from coffee containing one or morecompounds that elicit a bitter taste perception.

Further, the invention embraces the production of coffee and coffeeflavored foods, beverages and medicinals which have been treated toremove compounds that specifically activate these bitter tastereceptors, e.g., foods and beverages that have been processed to removeor reduce the amount of bitter compounds comprised therein.

In some aspects, the invention also relates to the structural classes ofcompounds represented by the two scaffolds given below. Scaffold 1 showsa representative urazole scaffold and scaffold 2 shows a representativehydantoin scaffold.

It is another specific object of the invention to use the compoundsshown supra and analogs of scaffold 1, and scaffold 2 as bitter blockersto reduce bitterness in food/pharmaceutical applications that aremediated by T2R8 receptors, especially coffee and coffee flavored foods,beverages and medicaments.

It is another object of the invention to confirm that the identifiedcompounds modulate, preferably inhibit or block, bitter taste, e.g. thatelicited by coffee and coffee flavored foods, beverages and medicamentsin human or animal taste tests, preferably human taste tests. Example 1shows representative sensory data for one of these compounds. The dataclearly demonstrates a significant decrease in bitterness for a specificT2R8 agonist and a significant increase in potency over a known T2R8bitter blocker.

It is another object of the invention to utilize compounds describedherein as additives or flavor modulators in compositions in order toinhibit or block the bitter taste elicited by compounds thatspecifically activate these taste receptors. A preferred object of theinvention is to use a compound that inhibits activation of T2R8receptors in order to block the bitter taste of compounds present incoffee and coffee flavored foods, beverages and medicinals.

Compounds identified according to the invention may be added to foods,beverages, cosmetics or medicinal compositions to modulate, preferablyblock bitter taste triggered by activation of hT2R8 by bitter compoundspresent in coffee and related foods, beverages and medicaments orstructurally related compounds or other bitter compounds, e.g.,compounds found in foods and beverages or medicinals or cosmetics thatelicit a bitter taste perception.

OBJECTS OF THE INVENTION

It is an object of the invention to provide assays that use hT2R8 and/orhT2R14 and chimeras and variants thereof which identify compounds andcompositions containing which elicit or block the bitter tasteassociated with coffee and coffee flavored foods, beverages andmedicaments.

It is a specific object of the invention to provide assays that identifycompounds which activate or which block or modulate the activationand/or binding of hT2R8 to compounds or compositions containingresponsible for coffee's bitter taste.

It is also a specific object of the invention to provide assays thatidentify compounds which activate or which block or modulate theactivation and/or binding of hT2R14 to compounds or compositionscontaining responsible for coffee's bitter taste.

It is another specific object of the invention to provide the specificcompounds identified using the inventive assays and compositionscontaining especially coffee and coffee flavored foods, beverages andmedicaments.

It is a specific object of the invention to provide the compounds shownbelow which are T2R8 and T2R14 antagonists that have been shown to blockbitterness of coffee.

It is another specific object of the invention to use the compoundsshown supra and analogs of Compound A, Compound B and Compound C asbitter blockers to reduce bitterness in food/pharmaceutical applicationthat is mediated by T2R8 and/or T2R14 receptors, especially coffee andcoffee flavored foods, beverages and medicaments.

It is another specific object of the invention to use Compound C andanalogs thereof as broadly acting bitter blockers to reduce bitternessin food/pharmaceutical application that is mediated by any of humanT2R3, 7, 10, 14, 16, 44, 51, 55, 61, 63, 64, 65, or 71 and/or humanT2R5, 9, 13, 54, 67 or 75, especially in foods, beverages andmedicaments containing multiple bitter compounds, bitter compounds thatinteract with multiple bitter taste receptors or compositions containingunknown bitter compounds or bitter compounds wherein their receptorspecificity is unknown.

It is another specific object of the invention to provide the compoundswhich can be represented by the following formulae.

In a first aspect, a compound of structural Formula (I) is provided:

or a salt, hydrate, solvate or N-oxide thereof wherein:

Ar¹ a five or six membered aryl, heteroaryl or cycloalkyl ring;

m is 0, 1, 2 or 3;

R¹ is SO₂; C═O; C═S; or C═NOR⁴;

X is selected from the group consisting of hydrogen, halogen, alkyl,substituted alkyl, aryl, substituted aryl, arylalkyl, substitutedarylalkyl, acyl, substituted acyl, heteroalkyl, substituted heteroalkyl,heteroaryl, substituted heteroaryl, heteroarylalkyl, substitutedheteroarylalkyl, CN, NO₂, OR⁶, S(O)_(b)R⁶, NR⁶R⁷, CONR⁶R⁷, CO₂R⁶,NR⁶CO₂R⁷, NR⁶CONR⁷R⁸, NR⁶CSNR⁷R⁸, NR⁶C(═NH)NR⁷R⁸, SO₂NR⁵R⁶, NR⁵SO₂R⁶,NR⁵SO₂NR⁶R⁷, B(OR⁵)(OR⁶), P(O)(OR⁵)(OR⁶), and P(O)(R⁵)(OR⁶);

each R¹′ is independently selected from the group consisting ofhydrogen, halogen, alkyl, substituted alkyl, aryl, substituted aryl,arylalkyl, substituted arylalkyl, acyl, substituted acyl, heteroalkyl,substituted heteroalkyl, heteroaryl, substituted heteroaryl,heteroarylalkyl, substituted heteroarylalkyl, CN, NO₂, OR⁶, S(O)_(b)R⁶,NR⁶R⁷, CONR⁶R⁷, CO₂R⁶, NR⁶CO₂R⁷, NR⁶CONR⁷R⁸, NR⁶CSNR⁷R⁸, NR⁶C(═NH)NR⁷R⁸,SO₂NR⁵R⁶, NR⁵SO₂R⁶, NR⁵SO₂NR⁶R⁷, B(OR⁶)OR⁶, P(O)(OR⁵)(OR⁶), andP(O)(R⁵)(OR⁶);

or alternatively, X and/or at least one R¹′ together with the atoms towhich they are bonded form an aryl, substituted aryl, heteroaryl,substituted heteroaryl, cycloalkyl, substituted cycloalkyl,cydloheteroalkyl or substituted cycloheteroalkyl ring where the ring isoptionally fused to another aryl, substituted aryl, heteroaryl,substituted heteroaryl, cycloalkyl, substituted cycloalkyl,cycloheteroalkyl or substituted cycloheteroalkyl ring;

R⁴-R⁸ are independently selected from the group consisting of hydrogen,alkyl, substituted alkyl, aryl, substituted aryl, arylalkyl, substitutedarylalkyl, heteroalkyl, substituted heteroalkyl, heteroaryl, substitutedheteroaryl, heteroarylalkyl and substituted heteroarylalkyl oralternatively, R⁵ and R⁶, R⁶ and R⁷, R⁷ and R⁸, together with the atomsto which they are bonded form a cycloheteroalkyl or substitutedcycloheteroalkyl ring;

A and B are independently selected from the group consisting ofhydrogen, alkyl, substituted alkyl, aryl, substituted aryl, arylalkyl,substituted arylalkyl, acyl, substituted acyl, heteroalkyl, substitutedheteroalkyl, heteroaryl, substituted heteroaryl, heteroarylalkyl,substituted heteroarylalkyl; and

b is 0, 1, or 2.

In some embodiments, A and B, together with the nitrogen atom to whichthey are attached, form a ring that can be fused with additionalsubstituted or unsubstituted rings and can comprise at least one doublebond. A non-limiting example of such a ring includes a group having theformula:

In a second aspect the invention provides compounds of structuralFormula (II) shown below:

or a salt, hydrate, solvate or N-oxide thereof wherein:

Ar¹, Ar² and Ar³ are independently a five or six membered aryl,heteroaryl, or cycloalkyl ring;

m is 0, 1, 2 or 3;

n and p are independently 0, 1, 2, 3 or 4;

r and t are independently 0, 1 or 2;

Y and Z are independently selected from the group consisting of CR⁶R⁷,C═O, C═S, C═NOR⁶, O, NR⁶, and S(O)_(b);

R¹ is selected from the group consisting of SO₂, C═O, C═S, and C═NOR⁴;

X may be selected from the group consisting of hydrogen, alkyl,substituted alkyl, aryl, substituted aryl, arylalkyl, substitutedarylalkyl, acyl, substituted acyl, heteroalkyl, substituted heteroalkyl,heteroaryl, substituted heteroaryl, heteroarylalkyl, substitutedheteroarylalkyl, CN, NO₂, —OR⁶, S(O)_(b)R⁶, NR⁶R⁷, CONR⁶R⁷, CO₂R⁶,NR⁶CO₂R⁷, NR⁶CONR⁷R⁸, NR⁶CSNR⁷R⁸, NR⁶C(═NH)NR⁷R⁸, SO₂NR⁵R⁶, NR⁵SO₂R⁶,NR⁵SO₂NR⁶R⁷, B(OR⁵)(OR⁶), P(O)(OR⁵)(OR⁶, and P(O)(R⁵)(OR⁶);

X is preferably selected from the group consisting of hydrogen,heteroalkyl, substituted heteroalkyl, heteroaryl, substitutedheteroaryl, heteroarylalkyl, substituted heteroarylalkyl, CN,S(O)_(b)R⁶, CONR⁶R⁷, —CO₂R⁶, SO₂NR⁵R⁶, NR⁵SO₂R⁶, NR⁵SO₂NR⁶R⁷,B(OR⁵)(OR⁶), P(O)(OR⁵)(OR⁶), and P(O)(R⁵)(OR⁶).

each R¹′ is independently selected from the group consisting ofhydrogen, halogen, alkyl, substituted alkyl, aryl, substituted aryl,arylalkyl, substituted arylalkyl, acyl, substituted acyl, heteroalkyl,substituted heteroalkyl, heteroaryl, substituted heteroaryl,heteroarylalkyl, substituted heteroarylalkyl, CN, NO₂, OR⁶, S(O)_(b)R⁶,NR⁶R⁷, CONR⁶R⁷, CO₂R⁶, NR⁶CO₂R⁷, NR⁶CONR⁷R⁸, NR⁶CSNR⁷R⁸, NR⁶C(═NH)NR⁷R⁸,SO₂NR⁵R⁶, NR⁵SO₂R⁶, NR⁵SO₂NR⁶R⁷, B(OR⁵)(OR⁶), P(O)(OR⁵)(OR⁶), andP(O)(R⁵)(OR⁶);

each R²′ is independently selected from the group consisting ofhydrogen, halogen, alkyl, substituted alkyl, aryl, substituted aryl,arylalkyl, substituted arylalkyl, acyl, substituted acyl, heteroalkyl,substituted heteroalkyl, heteroaryl, substituted heteroaryl,heteroarylalkyl, substituted heteroarylalkyl, CN, NO₂, OR⁶, S(O)_(b)R6,NR⁶R⁷, CONR⁶R⁷, CO₂R⁶, NR⁶CO₂R⁷, NR⁶CONR⁷R⁸, NR⁶CSNR⁷R⁸, NR⁶C(═NH)NR⁷R⁸,SO₂NR⁵R⁶, NR⁵SO₂R⁶, NR⁵SO₂NR⁶R⁷, B(OR⁵)(OR⁶), and P(O)(OR⁵)(OR⁶);

each R³′ is independently selected from the group consisting ofhydrogen, halogen, alkyl, substituted alkyl, aryl, substituted aryl,arylalkyl, substituted arylalkyl, acyl, substituted acyl, heteroalkyl,substituted heteroalkyl, heteroaryl, substituted heteroaryl,heteroarylalkyl, substituted heteroarylalkyl, CN, NO₂, OR⁶, S(O)_(b)R⁶,NR⁶R⁷, CONR⁶R⁷, CO₂R⁶, NR⁶CO₂R⁷, NR⁶CONR⁷R⁸, NR⁶CSNR⁷R⁸, NR⁶C(═NH)NR⁷R⁸,SO₂NR⁵R⁶, NR⁵SO₂R⁶, NR⁵SO₂NR⁶R⁷, B(OR⁵)(OR⁶), P(O)(OR⁵)(OR⁶), andP(O)(R⁵)(OR⁶);

or alternatively, X and/or at least one of R¹′ together with the atomsto which they are bonded form an aryl, substituted aryl, heteroaryl,substituted heteroaryl, cycloalkyl, substituted cycloalkyl,cycloheteroalkyl or substituted cycloheteroalkyl ring where the ring isoptionally fused to another aryl, substituted aryl, heteroaryl,substituted heteroaryl, cycloalkyl, substituted cycloalkyl,cycloheteroalkyl or substituted cycloheteroalkyl ring;

R⁴-R⁸ are independently hydrogen, alkyl, substituted alkyl, aryl,substituted aryl, arylalkyl, substituted arylalkyl, heteroalkyl,substituted heteroalkyl, heteroaryl, substituted heteroaryl,heteroarylalkyl or substituted heteroarylalkyl or alternatively, R⁵ andR⁶, R⁶ and R⁷, R⁷ and R⁸, together with the atoms to which they arebonded form a cycloheteroalkyl or substituted cycloheteroalkyl ring;

b is 0, 1, or 2.

In another aspect the invention provides compounds having structuralFormula (III) shown below:

or a salt, hydrate, solvate or N-oxide thereof wherein:

Ar¹, Ar² and Ar³ are independently a five or six membered aryl,heteroaryl, or cycloalkyl ring, and Ar² and Ar³ may optionally beomitted;

m is 0, 1, 2 or 3;

n and p are independently 0, 1, 2, 3 or 4;

each R¹′ is independently selected from the group consisting ofhydrogen, halogen, alkyl, substituted alkyl, aryl, substituted aryl,arylalkyl, substituted arylalkyl, acyl, substituted acyl, heteroalkyl,substituted heteroalkyl, heteroaryl, substituted heteroaryl,heteroarylalkyl, substituted heteroarylalkyl, CN, NO₂, OR⁶, S(O)_(b)R⁶,NR⁶R⁷, CONR⁶R⁷, CO₂R⁶, NR⁶CO₂R⁷, NR⁶CONR⁷R⁸, NR⁶CSNR⁷R⁸, NR⁶C(═NH)NR⁷R⁸,SO₂NR⁵R⁶, NR⁵SO₂R⁶, NR⁵SO₂NR⁶R⁷, B(OR⁵)(OR⁶), P(O)(OR⁵)(OR⁶), andP(O)(R⁵)(OR⁶);

each R²′ is independently selected from the group consisting ofhydrogen, halogen, alkyl, substituted alkyl, aryl, substituted aryl,arylalkyl, substituted arylalkyl, acyl, substituted acyl, heteroalkyl,substituted heteroalkyl, heteroaryl, substituted heteroaryl,heteroarylalkyl, substituted heteroarylalkyl, CN, NO₂, OR⁶, S(O)_(b)R⁶,NR⁶R⁷, CONR⁶R⁷, CO₂R⁶, NR⁶CO₂R⁷, NR⁶CONR⁷R⁸, NR⁶CSNR⁷R⁸, NR⁶C(═NH)NR⁷R⁸,SO₂NR⁵R⁶, NR⁵SO₂R⁶, NR⁵SO₂NR⁶R⁷, B(OR⁵)(OR⁶), P(O)(OR⁵)(OR⁶), andP(O)(R⁵)(OR⁶);

each R³′ is independently selected from the group consisting ofhydrogen, halogen, alkyl, substituted alkyl, aryl, substituted aryl,arylalkyl, substituted arylalkyl, acyl, substituted acyl, heteroalkyl,substituted heteroalkyl, heteroaryl, substituted heteroaryl,heteroarylalkyl, substituted heteroarylalkyl, CN, NO₂, OR⁶, S(O)_(b)R⁶,NR⁶R⁷, CONR⁶R⁷, CO₂R⁶, NR⁶CO₂R⁷, NR⁶CONR⁷R⁸, NR⁶CSNR⁷R⁸, NR⁶C(═NH)NR⁷R⁸,SO₂NR⁵R⁶, NR⁵SO₂R⁶, NR⁵SO₂NR⁶R⁷, B(OR⁵)(OR⁶), P(O)(OR⁵)(OR⁶), andP(O)(R⁵)(OR⁶);

R⁵-R⁸ are independently hydrogen, alkyl, substituted alkyl, aryl,substituted aryl, arylalkyl, substituted arylalkyl, heteroalkyl,substituted heteroalkyl, heteroaryl, substituted heteroaryl,heteroarylalkyl or substituted heteroarylalkyl or alternatively, R⁵ andR⁶, R⁶ and R⁷, R⁷ and R⁸, together with the atoms to which they arebonded form a cycloheteroalkyl or substituted cycloheteroalkyl ring;

b is 0, 1, or 2.

In yet another aspect the invention provides a compound having thestructure below:

or a salt, hydrate, solvate or N-oxide thereof.

In yet another aspect the invention provides compounds having thestructure below:

or a salt, hydrate, solvate or N-oxide thereof.

In still other embodiments, the invention provides compounds having thestructure below:

or a salt, hydrate, solvate or N-oxide thereof.

In a related aspect, a compound of structural Formula (IV) is provided:

or a salt, hydrate, solvate or N-oxide thereof wherein:

Ar⁴ and Ar⁵ are independently a five or six membered aryl or heteroarylring;

W is selected from the group consisting of CR⁶R⁷, C═O, C═S; C═NOR⁶; O,NR⁶, S, SO, SO₂, and (CH₂)_(n);

n is 0, 1, 2, or 3;

G is selected from the group consisting of CR⁶R⁷, C═O, C═S, C═NOR⁶, andS(O)_(b);

R²⁰ is selected from the group consisting of hydrogen, arylalkenyl,heteroarylalkenyl, arylalkyl, heteroarylalkyl, aryl, heteroaryl, alkyl,alkoxy-alkyl, alkenyl, cycloalkyl, cycloalkenyl, and substitutedderivatives;

R²¹ is selected from the group consisting of an arylalkenyl,heteroarylalkenyl, arylalkyl, heteroarylalkyl, aryl, heteroaryl, alkyl,alkoxy-alkyl, alkenyl, cycloalkyl, cycloalkenyl, and substitutedderivatives;

R⁶ and R⁷ are independently selected from the group consisting ofhydrogen, alkyl, substituted alkyl, aryl, substituted aryl, arylalkyl,substituted arylalkyl, heteroalkyl, substituted heteroalkyl, heteroaryl,substituted heteroaryl, heteroarylalkyl or substituted heteroarylalkylor alternatively, R⁶ and R⁷, together with the atoms to which they arebonded form a cycloheteroalkyl or substituted cycloheteroalkyl ring; and

b is 0, 1, or 2.

In another related aspect a compound of structural Formula (V) isprovided:

or a salt, hydrate, solvate or N-oxide thereof wherein:

Ar⁴ and Ar⁵ are independently a five or six membered aryl or heteroarylring;

n is 0, 1, 2, or 3;

R²¹ is selected from the group consisting of an arylalkenyl,heteroarylalkenyl, arylalkyl, hereoarylalky, aryl, heteroaryl, alkyl,alkoxy-alkyl, alkenyl, cycloalkyl, cycloalkenyl, and substitutedderivatives;

R³⁵ is selected from the group consisting of hydrogen, alkyl, andsubstituted alkyl.

In still additional embodiments the invention a compound of structuralFormula (VI) is provided

or a salt, hydrate, solvate or N-oxide thereof wherein:

R³⁰ is selected from the group consisting of an arylalkenyl,heteroarylalkenyl, arylalkyl, hereoarylalky, aryl, heteroaryl, alkyl,alkoxy-alkyl, alkenyl, cycloalkyl, cycloalkenyl, and substitutedderivatives;

R³⁵ is selected from the group consisting of hydrogen, alkyl, andsubstituted alkyl.

In still additional embodiments the invention provides compounds havingthe structure below:

or a salt, hydrate, solvate or N-oxide thereof,wherein each R is independently Cl, MeO, CN, EtO, OH, Me, —SO₂Me, F, orH, andn is 0, 1, 2, 3 or 4.

In still other embodiments, the invention provides compounds having thestructure below:

or a salt, hydrate, solvate or N-oxide thereof,wherein each R is independently MeO or OH andn is 0, 1, 2, 3 or 4.

In still other embodiments, the invention provides compounds having thestructure below:

or a salt, hydrate, solvate or N-oxide thereof,wherein R is H, Me, Et, OCOMe, CH₂OH, OMe, or Ph.

In still other embodiments, the invention provides compounds having thestructure below:

or a salt, hydrate, solvate or N-oxide thereof.

In still other embodiments, the invention provides compounds having thestructure below:

or a salt, hydrate, solvate or N-oxide thereof.

In still other embodiments, the invention provides compounds having thestructure below:

or a salt, hydrate, solvate or N-oxide thereof.

In still other embodiments, the invention provides compounds having thestructure below:

or a salt, hydrate, solvate or N-oxide thereof.

In one aspect, the invention relates to a compound of the formula:

-   -   or a salt, hydrate, solvate, N-oxide or prodrug thereof,    -   wherein Ar⁶ and Ar⁷ are, the same or different independently one        from the other, a five- or six-membered aryl group or a five- or        six-membered heteroaryl group;    -   Alk is an alkyl group, optionally interrupted by a heteroatom;    -   R₃₆ and R₃₇ are, the same or different independently one from        the other, H, alkyl, or, R₃₆ and R₃₇, together with the atoms to        which they are attached, form an optionally substituted five- or        six-membered heterocycle; and    -   R₃₈ is H, substituted or unsubstituted alkyl, substituted or        unsubstituted cycloalkyl alkyl, substituted or unsubstituted        heterocycloalkylalkyl, substituted or unsubstituted aryl,        substituted or unsubstituted arylamidoalkyl, substituted or        unsubstituted heteroarylamidoalkyl, substituted or unsubstituted        aryl alkyl, substituted or unsubstituted arylalkoxy, substituted        or unsubstituted heteroaryl, substituted or unsubstituted        heteroarylalkyl, or haloalkyl.

In one aspect the compounds of the invention contain a five-memberedheterocycle. In one embodiment, the five-membered heterocycle is ahydantoin or a substituted or unsubstituted cyclic urea.

In one embodiment, the hydantoin is a hydantoin of the formula:

-   -   or a salt, hydrate, solvate, N-oxide or prodrug thereof,    -   wherein Ar⁶ and Ar⁷ are, the same or different independently one        from the other, a five- or six-membered aryl group or a five- or        six-membered heteroaryl group;    -   Alk is an alkyl group, optionally interrupted by a heteroatom;    -   R₃₈ is H, substituted or unsubstituted alkyl, substituted or        unsubstituted cycloalkyl alkyl, substituted or unsubstituted        heterocycloalkylalkyl, substituted or unsubstituted aryl,        substituted or unsubstituted arylamidoalkyl, substituted or        unsubstituted heteroarylamidoalkyl, substituted or unsubstituted        arylalkyl, substituted or unsubstituted arylalkoxy, substituted        or unsubstituted heteroaryl, substituted or unsubstituted        heteroarylalkyl, or haloalkyl; and    -   R₃₉ and R₄₀ are, the same or different independently one from        the other, H, substituted or unsubstituted alkyl, substituted or        unsubstituted cycloalkyl alkyl, substituted or unsubstituted        heterocycloalkylalkyl, substituted or unsubstituted aryl,        substituted or unsubstituted arylamidoalkyl, substituted or        unsubstituted arylalkylamidoalkyl, substituted or unsubstituted        heteroarylamidoalkyl, substituted or unsubstituted        heteroarylalkylamidoalkyl, substituted or unsubstituted        arylalkyl, substituted or unsubstituted arylalkoxy, substituted        or unsubstituted heteroaryl, substituted or unsubstituted        heteroarylalkyl, haloalkyl, or R₃₉ and R₄₀, together with the        carbon atom to which they are attached, form a C═O group or a        substituted or unsubstituted alkenyl group.

In another aspect the compounds of the invention contain a five-memberedheterocycle which is a urazole. In one embodiment, the urazole is aurazole of the formula:

-   -   or a salt, hydrate, solvate, N-oxide or prodrug thereof,    -   wherein Ar⁶ and Ar⁷ are, the same or different independently one        from the other, a five- or six-membered aryl group or a five- or        six-membered heteroaryl group;    -   Alk is an alkyl group, optionally interrupted by a heteroatom;    -   R₃₈ is H, substituted or unsubstituted alkyl, substituted or        unsubstituted cycloalkyl alkyl, substituted or unsubstituted        heterocycloalkylalkyl, substituted or unsubstituted aryl,        substituted or unsubstituted arylamidoalkyl, substituted or        unsubstituted heteroarylamidoalkyl, substituted or unsubstituted        arylalkyl, substituted or unsubstituted arylalkoxy, substituted        or unsubstituted heteroaryl, substituted or unsubstituted        heteroarylalkyl, or haloalkyl; and    -   R₄₁ is H, substituted or unsubstituted alkyl, substituted or        unsubstituted cycloalkyl alkyl, substituted or unsubstituted        heterocycloalkylalkyl, substituted or unsubstituted aryl,        substituted or unsubstituted arylamidoalkyl, substituted or        unsubstituted arylalkylamidoalkyl, substituted or unsubstituted        heteroarylamidoalkyl, substituted or unsubstituted        heteroarylalkylamidoalkyl, substituted or unsubstituted        arylalkyl, substituted or unsubstituted arylalkoxy, substituted        or unsubstituted heteroaryl, substituted or unsubstituted        heteroarylalkyl, or haloalkyl.

In another aspect the compounds of the invention contain a six-memberedheterocycle. In one embodiment, the six-membered heterocycle is asix-membered heterocycle of the formula:

-   -   or a salt, hydrate, solvate, N-oxide or prodrug thereof,    -   wherein R₃₈ is H, substituted or unsubstituted alkyl,        substituted or unsubstituted cycloalkyl alkyl, substituted or        unsubstituted heterocycloalkylalkyl, substituted or        unsubstituted aryl, substituted or unsubstituted arylamidoalkyl,        substituted or unsubstituted heteroarylamidoalkyl, substituted        or unsubstituted arylalkyl, substituted or unsubstituted        arylalkoxy, substituted or unsubstituted heteroaryl, substituted        or unsubstituted heteroarylalkyl, or haloalkyl; and    -   R₄₂, R₄₃, R₄₄, R₄₅, and R₄₆ are, the same or different        independently one from the other, H, substituted or        unsubstituted alkyl, substituted or unsubstituted cycloalkyl        alkyl, substituted or unsubstituted arylalkyl, substituted or        unsubstituted arylalkoxy, substituted or unsubstituted        heteroaryl, substituted or unsubstituted heteroarylalkyl, or R₄₂        and R₄₃, or R₄₅ and R₄₆, together with the carbon atoms to which        each are attached, form a C═O group.

In another aspect, the invention relates to a compound of the formula:

-   -   or a salt, hydrate, solvate, N-oxide or prodrug thereof,    -   wherein Alk is an alkyl group, optionally interrupted by a        heteroatom;    -   M¹ is N or CR₄₉, wherein R₄₉ is H or substituted or        unsubstituted alkyl;    -   M² is N or CR₅₀, wherein R₅₀ is H or substituted or        unsubstituted alkyl;    -   R₃₆ and R₃₇ are, the same or different independently one from        the other, H, alkyl, or, R₃₆ and R₃₇, together with the atoms to        which they are attached, form an optionally substituted five- or        six-membered heterocycle; and    -   R₃₈ is H, substituted or unsubstituted alkyl, substituted or        unsubstituted cycloalkyl alkyl, substituted or unsubstituted        heterocycloalkylalkyl, substituted or unsubstituted aryl,        substituted or unsubstituted arylamidoalkyl, substituted or        unsubstituted heteroarylamidoalkyl, substituted or unsubstituted        arylalkyl, substituted or unsubstituted arylalkoxy, substituted        or unsubstituted heteroaryl, substituted or unsubstituted        heteroarylalkyl, or haloalkyl;    -   R₄₇ is H, substituted or unsubstituted alkyl, substituted or        unsubstituted alkoxy, substituted or unsubstituted aryl,        substituted or unsubstituted arylalkyl, or halo; and    -   R₄₈ is H, substituted or unsubstituted alkyl, substituted or        unsubstituted alkoxy, substituted or unsubstituted aryl,        substituted or unsubstituted arylalkyl, or halo.

In still another aspect, the invention relates to a compound of theformula:

-   -   or a salt, hydrate, solvate, N-oxide or prodrug thereof,    -   wherein Alk is an alkyl group, optionally interrupted by a        heteroatom;    -   T₁ is C═O and Q is CR₅₁R₅₂ or NR₅₁, wherein R₅₁ and R₅₂ are, the        same or different independently one from the other, H,        substituted or unsubstituted alkyl, substituted or unsubstituted        cycloalkyl alkyl, substituted or unsubstituted        heterocycloalkylalkyl, substituted or unsubstituted aryl,        substituted or unsubstituted arylamidoalkyl, substituted or        unsubstituted aryl alkylamidoalkyl, substituted or unsubstituted        heteroarylamidoalkyl, substituted or unsubstituted        heteroarylalkylamidoalkyl, substituted or unsubstituted        arylalkyl, substituted or unsubstituted arylalkoxy, substituted        or unsubstituted heteroaryl, substituted or unsubstituted        heteroarylalkyl, haloalkyl, or R₅₁ and R₅₂, together with the        carbon atom to which they are attached, form a C═O group or a        substituted or unsubstituted alkenyl group;    -   M¹ is N or CR₄₉, wherein R₄₉ is H or substituted or        unsubstituted alkyl;    -   M² is N or CR₅₀, wherein R₅₀ is H or substituted or        unsubstituted alkyl;    -   R₃₈ is H, substituted or unsubstituted alkyl, substituted or        unsubstituted cycloalkyl alkyl, substituted or unsubstituted        heterocycloalkylalkyl, substituted or unsubstituted aryl,        substituted or unsubstituted arylamidoalkyl, substituted or        unsubstituted heteroarylamidoalkyl, substituted or unsubstituted        arylalkyl, substituted or unsubstituted arylalkoxy, substituted        or unsubstituted heteroaryl, substituted or unsubstituted        heteroarylalkyl, or haloalkyl;    -   R₄₇ is H, substituted or unsubstituted alkyl, substituted or        unsubstituted alkoxy, substituted or unsubstituted aryl,        substituted or unsubstituted arylalkyl, or halo; and    -   R₄₈ is H, substituted or unsubstituted alkyl, substituted or        unsubstituted alkoxy, substituted or unsubstituted aryl,        substituted or unsubstituted arylalkyl, or halo.

In another embodiment, the invention relates to a compound of theformula:

or a salt, hydrate, solvate, N-oxide or prodrug thereof.

In another aspect, the invention relates to a compound of the formula:

or a salt, hydrate, solvate, N-oxide or prodrug thereof.

In still another aspect, the invention relates to a method of making acompound of the formula:

-   -   or a salt, hydrate, solvate, N-oxide or prodrug thereof,    -   wherein Alk is an alkyl group, optionally interrupted by a        heteroatom;    -   T₂ is C═S, C═O, or S(O)₂;    -   R₅₃ is substituted or unsubstituted alkyl, substituted or        unsubstituted cycloalkyl, substituted or unsubstituted        heteroaryl, substituted or unsubstituted aryl, or substituted or        unsubstituted arylalkyl;    -   M¹ is N or CR₅₄, wherein R₅₄ is H or substituted or        unsubstituted alkyl;    -   M² is N or CR₅₅, wherein R₅₅ is H or substituted or        unsubstituted alkyl;    -   R₅₆ is H, substituted or unsubstituted alkyl, substituted or        unsubstituted alkoxy, substituted or unsubstituted aryl,        substituted or unsubstituted arylalkyl, or halo; and    -   R₅₇ is H, substituted or unsubstituted alkyl, substituted or        unsubstituted alkoxy, substituted or unsubstituted aryl,        substituted or unsubstituted arylalkyl, or halo;    -   wherein the method comprises reacting a compound of the formula:

-   -   wherein R₅₆, R₅₇, and Alk are defined above and J is a leaving        group;    -   with a compound of the formula:

-   -   wherein M¹ and M² are defined above to give a compound of the        formula

-   -   having an NO₂ group;    -   reducing the NO₂ group to give a compound having an NH₂ group;        and    -   reacting the compound having an NH₂ group with a compound of the        formula

-   -   wherein J₂ is a leaving group and T₂ and R₅₃ are defined above.

In still another aspect, the invention relates to a method of making acompound of the formula:

-   -   or a salt, hydrate, solvate, N-oxide or prodrug thereof,    -   wherein Alk is an alkyl group, optionally interrupted by a        heteroatom;    -   R₅₁ and R₅₂ are, the same or different independently one from        the other, H, substituted or unsubstituted alkyl, substituted or        unsubstituted cycloalkyl alkyl, substituted or unsubstituted        heterocycloalkylalkyl, substituted or unsubstituted aryl,        substituted or unsubstituted arylamidoalkyl, substituted or        unsubstituted arylalkylamidoalkyl, substituted or unsubstituted        heteroarylamidoalkyl, substituted or unsubstituted        heteroarylalkylamidoalkyl, substituted or unsubstituted        arylalkyl, substituted or unsubstituted arylalkoxy, substituted        or unsubstituted heteroaryl, substituted or unsubstituted        heteroarylalkyl, haloalkyl, or R₅₁ and R₅₂, together with the        carbon atom to which they are attached, form a substituted or        unsubstituted alkenyl group;    -   M¹ is N or CR₄₉, wherein R₄₉ is H or substituted or        unsubstituted alkyl;    -   M² is N or CR₅₀, wherein R₅₀ is H or substituted or        unsubstituted alkyl;    -   R₃₈ is H, substituted or unsubstituted alkyl, substituted or        unsubstituted cycloalkyl alkyl, substituted or unsubstituted        heterocycloalkylalkyl, substituted or unsubstituted aryl,        substituted or unsubstituted arylamidoalkyl, substituted or        unsubstituted heteroarylamidoalkyl, substituted or unsubstituted        arylalkyl, substituted or unsubstituted arylalkoxy, substituted        or unsubstituted heteroaryl, substituted or unsubstituted        heteroarylalkyl, or haloalkyl;    -   R₄₇ is H, substituted or unsubstituted alkyl, substituted or        unsubstituted alkoxy, substituted or unsubstituted aryl,        substituted or unsubstituted arylalkyl, or halo; and    -   R₄₈ is H, substituted or unsubstituted alkyl, substituted or        unsubstituted alkoxy, substituted or unsubstituted aryl,        substituted or unsubstituted arylalkyl, or halo;    -   wherein the method comprises heating a compound of the formula:

-   -   wherein R₄₇, R₄₈, Alk, M¹, and M² are defined above;    -   to convert the —CON₃ group to a —N═C═O group, and then reacting        with a compound of the formula:

-   -   wherein J₃ is a leaving group and R₃₈, R₅₁, and R₅₂ are defined        above.

In still another aspect, the invention relates to a method of making acompound of the formula:

-   -   or a salt, hydrate, solvate, N-oxide or prodrug thereof,    -   wherein Alk is an alkyl group, optionally interrupted by a        heteroatom;    -   R₅₂ is H, substituted or unsubstituted alkyl, substituted or        unsubstituted cycloalkyl alkyl, substituted or unsubstituted        heterocycloalkylalkyl, substituted or unsubstituted aryl,        substituted or unsubstituted arylamidoalkyl, substituted or        unsubstituted arylalkylamidoalkyl, substituted or unsubstituted        heteroarylamidoalkyl, substituted or unsubstituted        heteroarylalkylamidoalkyl, substituted or unsubstituted        arylalkyl, substituted or unsubstituted arylalkoxy, substituted        or unsubstituted heteroaryl, substituted or unsubstituted        heteroarylalkyl, haloalkyl;    -   M¹ is N or CR₄₉, wherein R₄₉ is H or substituted or        unsubstituted alkyl;    -   M² is N or CR₅₀, wherein R₅₀ is H or substituted or        unsubstituted alkyl;    -   R₃₈ is H, substituted or unsubstituted alkyl, substituted or        unsubstituted cycloalkyl alkyl, substituted or unsubstituted        heterocycloalkylalkyl, substituted or unsubstituted aryl,        substituted or unsubstituted arylamidoalkyl, substituted or        unsubstituted heteroarylamidoalkyl, substituted or unsubstituted        arylalkyl, substituted or unsubstituted arylalkoxy, substituted        or unsubstituted heteroaryl, substituted or unsubstituted        heteroarylalkyl, or haloalkyl;    -   R₄₇ is H, substituted or unsubstituted alkyl, substituted or        unsubstituted alkoxy, substituted or unsubstituted aryl,        substituted or unsubstituted arylalkyl, or halo; and    -   R₄₈ is H, substituted or unsubstituted alkyl, substituted or        unsubstituted alkoxy, substituted or unsubstituted aryl,        substituted or unsubstituted arylalkyl, or halo;    -   wherein the method comprises heating a compound of the formula:

-   -   wherein R₄₇, R₄₈, Alk, M¹, and M² are defined above;    -   to convert the —CON₃ group to a —N═C═O group, and then reacting        with a hydrazine of the formula:

-   -   wherein R₃₈ is defined above.

In still another aspect, the invention relates to a method of making acompound of the formula:

-   -   or a salt, hydrate, solvate or N-oxide thereof wherein:    -   Ar¹, Ar² and Ar³ are independently a five or six membered aryl,        heteroaryl, or cycloalkyl ring, and Ar² and Ar³ may optionally        be omitted;    -   m is 0, 1, 2 or 3;    -   n and p are independently 0, 1, 2, 3 or 4;    -   each R¹′ is independently selected from the group consisting of        hydrogen, halogen, alkyl, substituted alkyl, aryl, substituted        aryl, arylalkyl, substituted arylalkyl, acyl, substituted acyl,        heteroalkyl, substituted heteroalkyl, heteroaryl, substituted        heteroaryl, heteroarylalkyl, substituted heteroarylalkyl, CN,        NO₂, OR⁶, S(O)_(b)R⁶, NR⁶R⁷, CONR⁶R⁷, CO₂R⁶, NR⁶CO₂R⁷,        NR⁶CONR⁷R⁸, NR⁶CSNR⁷R⁸, NR⁶C(═NH)NR⁷R⁸, SO₂NR⁵R⁶, NR⁵SO₂R⁶,        NR⁵SO₂NR⁶R⁷, B(OR⁵)(OR⁶), P(O)(OR⁵)(OR⁶), and P(O)(R⁵)(OR⁶);    -   each R²′ is independently selected from the group consisting of        hydrogen, halogen, alkyl, substituted alkyl, aryl, substituted        aryl, arylalkyl, substituted arylalkyl, acyl, substituted acyl,        heteroalkyl, substituted heteroalkyl, heteroaryl, substituted        heteroaryl, heteroarylalkyl, substituted heteroarylalkyl, CN,        NO₂, OR⁶, S(O)_(b)R⁶, NR⁶R⁷, CONR⁶R⁷, CO₂R⁶, NR⁶CO₂R⁷,        NR⁶CONR⁷R⁸, NR⁶CSNR⁷R⁸, NR⁶C(═NH)NR⁷R⁸, SO₂NR⁵R⁶, NR⁵SO₂R⁶,        NR⁵SO₂NR⁶R⁷, B(OR⁵)(OR⁶), P(O)(OR⁵)(OR⁶), and P(O)(R⁵)(OR⁶);    -   each R³′ is independently selected from the group consisting of        hydrogen, halogen, alkyl, substituted alkyl, aryl, substituted        aryl, arylalkyl, substituted arylalkyl, acyl, substituted acyl,        heteroalkyl, substituted heteroalkyl, heteroaryl, substituted        heteroaryl, heteroarylalkyl, substituted heteroarylalkyl, CN,        NO₂, OR⁶, S(O)_(b)R⁶, NR⁶R⁷, CONR⁶R⁷, CO₂R⁶, NR⁶CO₂R⁷,        NR⁶CONR⁷R⁸, NR⁶CSNR⁷R⁸, NR⁶C(═NH)NR⁷R⁸, SO₂NR⁵R⁶, NR⁵SO₂R⁶,        NR⁵SO₂NR⁶R⁷, B(OR⁵)(OR⁶), P(O)(OR⁵)(OR⁶), and P(O)(R⁵)(OR⁶);    -   R⁵-R⁸ are independently hydrogen, alkyl, substituted alkyl,        aryl, substituted aryl, arylalkyl, substituted arylalkyl,        heteroalkyl, substituted heteroalkyl, heteroaryl, substituted        heteroaryl, heteroarylalkyl or substituted heteroarylalkyl or        alternatively, R⁶ and R⁷, R⁷ and R⁸, together with the atoms to        which they are bonded form a cycloheteroalkyl or substituted        cycloheteroalkyl ring;    -   b is 0, 1, or 2;    -   wherein the method comprises reacting a compound of the formula:

-   -   wherein J is a leaving group;    -   with a compound of the formula:

-   -   to give a product; and    -   reacting the product with a compound of the formula:

-   -   wherein J is a leaving group.

In still another aspect, the invention relates to a compound of theformula:

-   -   or a salt, hydrate, solvate or N-oxide thereof,    -   wherein Ar⁶ and Ar⁷ are, the same or different independently one        from the other, a five- or six-membered aryl group or a five- or        six-membered heteroaryl group;    -   Alk is an alkyl group, optionally interrupted by a heteroatom;    -   R₅₈ is H, alkyl, substituted alkyl, aryl, substituted aryl,        arylalkyl, substituted arylalkyl, acyl, substituted acyl,        heteroalkyl, substituted heteroalkyl, heteroaryl, substituted        heteroaryl, heteroarylalkyl, or substituted heteroarylalkyl;    -   R₅₉ and R₆₀ are the same or different independently one from the        other, H, alkyl, substituted alkyl, aryl, substituted aryl,        arylalkyl, substituted arylalkyl, acyl, substituted acyl,        heteroalkyl, substituted heteroalkyl, heteroaryl, substituted        heteroaryl, heteroarylalkyl or substituted heteroarylalkyl, with        the proviso that when one of R₅₉ or R₆₀ is H, then the other of        R₅₉ or R₆₀ is not H; or    -   R₅₉ and R₆₀, together with the atom to which they are attached        form a cycloalkyl, a substituted cycloalkyl, a cycloheteroalkyl        or substituted cycloheteroalkyl ring; or    -   R₅₈ and R₆₀, together with the atoms to which they are attached,        form a five- or six-membered cycloheteroalkyl or substituted        cycloheteroalkyl ring.

In another aspect, the invention relates to a compound of the formula:

-   -   a salt, hydrate, solvate or N-oxide thereof,    -   wherein:    -   R₆₁ is H, alkyl, substituted alkyl, aryl, substituted aryl,        arylalkyl, substituted arylalkyl, acyl, substituted acyl,        heteroalkyl, substituted heteroalkyl, heteroaryl, substituted        heteroaryl, heteroarylalkyl or substituted heteroarylalkyl;    -   R₆₂ and R₆₃ are the same or different independently one from the        other, H, alkyl, substituted alkyl, aryl, substituted aryl,        arylalkyl, substituted arylalkyl, acyl, substituted acyl,        heteroalkyl, substituted heteroalkyl, heteroaryl, substituted        heteroaryl, heteroarylalkyl or substituted heteroarylalkyl, with        the proviso that when one of R₆₂ or R₆₃ is H, then the other of        R₆₂ or R₆₃ is not H;    -   or R₆₂ and R₆₃, together with the atoms to which they are        attached form a cycloalkyl, a substituted cycloalkyl, a        cycloheteroalkyl or substituted cycloheteroalkyl ring; or    -   R₆₁ and R₆₂, together with the atoms to which they are attached,        form a five- or six-membered cycloheteroalkyl or substituted        cycloheteroalkyl ring.

In yet another aspect, the invention relates to a compound of theformula:

-   -   a salt, hydrate, solvate or N-oxide thereof,    -   wherein:    -   R₆₄ and R₆₅ are the same or different independently one from the        other, H, alkyl, substituted alkyl, aryl, substituted aryl,        arylalkyl, substituted arylalkyl, acyl, substituted acyl,        heteroalkyl, substituted heteroalkyl, heteroaryl, substituted        heteroaryl, heteroarylalkyl or substituted heteroarylalkyl, with        the proviso that when one of R₆₄ or R₆₅ is H, then the other of        R₆₄ or R₆₅ is not H;    -   or R₆₄ and R₆₅, together with the atoms to which they are        attached, form a cycloalkyl, a substituted cycloalkyl, a        cycloheteroalkyl or substituted cycloheteroalkyl ring;    -   each R₆₆ is independently hydrogen, alkyl, substituted alkyl,        aryl, substituted aryl, arylalkyl, substituted arylalkyl, acyl,        substituted acyl, heteroalkyl, substituted heteroalkyl,        heteroaryl, substituted heteroaryl, heteroarylalkyl, substituted        heteroarylalkyl, F, Cl, —CN, —NO₂, —OR₆₇, —S(O)_(i)R₆₇,        —NR₆₇R₆₈, —CONR₆₇R₆₈, —CO₂R₆₇, —NR₆₇CO₂R⁶⁸, —NR₆₇CONR₆₈R₆₉,        —NR₆₇CSNR₆₈R₆₉ or —NR₆₇C(═NH)NR₆₈R₆₉, —SO₂NR₆₇R₆₈, —NR₆₇SO₂R₆₈,        —NR₆₇SO₂NR₆₈R₆₉, —B(OR₆₇)(OR₆₈), —P(O)(OR₆₇)(OR₆₈), or        —P(O)(R₆₇)(OR₆₈), wherein R₆₇, R₆₈, and R₆₉ are independently        hydrogen, alkyl, substituted alkyl, aryl, substituted aryl,        arylalkyl, substituted arylalkyl, heteroalkyl, substituted        heteroalkyl, heteroaryl, substituted heteroaryl, heteroarylalkyl        or substituted heteroarylalkyl or alternatively, R₆₇ and R₆₈ or        R₆₈ and R₆₉, together with the atoms to which they are attached        form a cycloheteroalkyl or substituted cycloheteroalkyl ring;    -   Ar¹⁰ is an aryl or heteroaryl ring;    -   g is 0, 1, 2, 3 or 4;    -   h is 0, 1, 2, 3 or 4; and    -   i is 0, 1 or 2.

In another aspect, the invention relates to a A compound of the formula:

-   -   a salt, hydrate, solvate or N-oxide thereof,    -   wherein:    -   each R₇₀ is independently hydrogen, alkyl, substituted alkyl,        aryl, substituted aryl, arylalkyl, substituted arylalkyl, acyl,        substituted acyl, heteroalkyl, substituted heteroalkyl,        heteroaryl, substituted heteroaryl, heteroarylalkyl, substituted        heteroarylalkyl, F, Cl, —CN, —NO₂, —OR₆₇, —S(O)_(i)R₆₇,        —NR₆₇R₆₈, —CONR₆₇R₆₈, —CO₂R₆₇, —NR₆₇CO₂R₆₈, —NR₆₇CONR₆₈R₆₉,        —NR₆₇CSNR₆₈R₆₉ or —NR₆₇C(═NH)NR₆₈R₆₉, —SO₂NR₆₇R₆₈, —NR₆₇SO₂R₆₈,        —NR₆₇SO₂NR₆₈R₆₉, —B(OR₆₇)(OR₆₈), —P(O)(OR₆₇(OR₆₈), or        —P(O)(R₆₇)(OR₆₈), wherein R₆₇, R₆₈, and R₆₉ are independently        hydrogen, alkyl, substituted alkyl, aryl, substituted aryl,        arylalkyl, substituted arylalkyl, heteroalkyl, substituted        heteroalkyl, heteroaryl, substituted heteroaryl, heteroarylalkyl        or substituted heteroarylalkyl or alternatively, R₆₇ and R₆₈ or        R₆₈ and R₆₉, together with the atoms to which they are attached        form a cycloheteroalkyl or substituted cycloheteroalkyl ring;    -   j is 0, 1 or 2;    -   k is 0, 1 or 2; and    -   l is 0, 1 or 2;    -   with the proviso that j and k are not simultaneously both 0.

It is another object of the invention to utilize compounds identified inthe assays described herein as additives or flavor modulators incompositions in order to inhibit or block the bitter taste elicited bycompounds that specifically activate these taste receptors. A preferredobject of the invention is to use a compound that inhibits activation ofat least one of the above-identified human T2R receptors in order toblock the bitter taste of compounds present in coffee and coffeeflavored foods, beverages and medicinals.

It is another object of the invention to utilize compounds of thepresent invention as broadly acting bitter blockers in order to inhibitor block the bitter taste elicited by compounds that specificallyactivate hT2R8 taste receptors, ligands that activate multiple bittertaste receptors, bitter compounds having unknown receptor specificity orcompositions containing unknown or multiple bitter compounds. In oneembodiment, the compounds of the invention are utilized to inhibitactivation of at least one of the above-identified human T2R receptorsthereby blocking the bitter taste of compounds present in coffee andcoffee flavored foods, beverages and medicinals. It is another object ofthe invention to confirm that the identified compounds modulate,preferably inhibit or block, bitter taste, e.g. that elicited by coffeeand coffee flavored foods, beverages and medicaments in human or animaltaste tests, preferably human taste tests.

It is another object of the invention to utilize compounds identified inthe assays described herein as additives or flavor modulators incompositions in order to inhibit or block the bitter taste elicited bycompounds that specifically activate these taste receptors. A preferredobject of the invention is to use a compound that inhibits activation ofat least one of the above-identified human T2R receptors in order toblock the bitter taste of compounds present in coffee and coffeeflavored foods, beverages and medicinals.

In an especially preferred embodiment Compound C and analogs thereof areused as broadly acting bitter blockers in order to inhibit or block thebitter taste elicited by compounds that specifically activate hT2R3, 7,10, 14, 16, 44, 51, 55, 61, 63, 64, 65, 71 and/or hT2R5, 9, 13, 54, 67and 75 taste receptors, ligands that activate multiple bitter tastereceptors, bitter compounds having unknown receptor specificity orcompositions containing unknown or multiple bitter compounds. GivenCompound C's broad antagonistic properties it should substantiallyalleviate bitter taste of most bitter compounds and compositionscontaining. A preferred object of the invention is to use a compoundthat inhibits activation of at least one of the above-identified humanT2R receptors such as Compound C or an analog in order to block thebitter taste of compounds present in coffee and coffee flavored foods,beverages and medicinals.

DETAILED DESCRIPTION OF THE FIGURES

FIG. 1 relates to experiments wherein a partially purified bitterfraction from coffee is used to screen the 25 human T2Rs in transientlytransfected HEK cells as described in Applicant's previous patentapplications incorporated by reference herein. As shown in FIG. 1, thecoffee fraction activated HEK293 cells transiently transfected withhT2R8 and hT2R14 in calcium imaging assay. A blue dye FD&C was used toreduce the fluorescence level of the coffee fraction, which wouldinterfere with the assay.

FIG. 2 is a plot of ΔF/F vs. log coffee fraction showing thedose-dependent response of hT2R8 and hT2R14 to a bitter tasting fractionderived from coffee. The assay was carried out using hT2R8 and hT2R14stable cell lines and automated fluorescence detector FLIPR.

FIG. 3 is a plot of percent inhibition of hT2R8 activity vs. log of theconcentration of a compound and shows the dose-dependent inhibition forcompounds A and B on a stable hT2R8 expressing cell line.

FIG. 4 is a plot of percent inhibition of hT2R14 activity vs. log of theconcentration of compound C and shows the dose inhibition for compound Con a stable hT2R8 expressing cell line.

FIG. 5 shows the inhibitory activity of Compound C against different (2)human bitter taste receptors.

FIG. 6 is a plot of receptor activity as a function of log of thesaccharin concentration. FIG. 6 shows the dose-response relationshipsand the effects of saccharin on receptor activities in transfected cellsexpressing variants of hT2R43, hT2R44 and hT2R8. hT2R8 is lessresponsive to saccharin in the in vitro assay than the “taster”hT2R43-W35 and hT2R44-W35 alleles, but responds better than the“non-taster” hT2R43-S35 and hT2R44-R35 alleles.

DETAILED DESCRIPTION OF THE INVENTION

According to the present invention, the compounds of the presentinvention can be used alleviate or reduce the bitter taste ofcompositions, e.g., an ingestible composition. As used herein, an“ingestible composition” includes any substance intended for oralconsumption either alone or together with another substance. Theingestible composition includes both “food or beverage products” and“non-edible products”. By “food or beverage products”, it is meant anyedible product intended for consumption by humans or animals, includingsolids, semi-solids, or liquids (e.g., beverages). The term “non-edibleproducts” or “noncomestible composition” includes supplements,nutraceuticals, functional food products (e.g., any fresh or processedfood claimed to have a health-promoting and/or disease-preventingproperties beyond the basic nutritional function of supplyingnutrients), pharmaceutical and over the counter medications, oral careproducts such as dentifrices and mouthwashes, cosmetic products such aslip balms and other personal care products.

The ingestible composition also includes pharmaceutical, medicinal orcomestible composition, or alternatively in a formulation, e.g., apharmaceutical or medicinal formulation or a food or beverage product orformulation.

The compounds of the present invention can also be provided,individually or in combination, with any ingestible composition known orlater discovered. For example, the ingestible composition can be acomestible composition or noncomestible composition. By “comestiblecomposition”, it is meant any composition that can be consumed as foodby humans or animals, including solids, gel, paste, foamy material,semi-solids, liquids, or mixtures thereof. By “noncomestiblecomposition”, it is meant any composition that is intended to beconsumed or used by humans or animals not as food, including solids,gel, paste, foamy material, semi-solids, liquids, or mixtures thereof.The noncomestible composition includes, but is not limited to medicalcomposition, which refers to a noncomestible composition intended to beused by humans or animals for therapeutic purposes. By “animal”, itincludes any non-human animal, such as, for example, farm animals andpets.

In one embodiment, the compounds of the invention can be added to anoncomestible composition or non-edible product, such as supplements,nutraceuticals, functional food products (e.g., any fresh or processedfood claimed to have a health-promoting and/or disease-preventingproperties beyond the basic nutritional function of supplyingnutrients), pharmaceutical and over the counter medications, oral careproducts such as dentifrices and mouthwashes, cosmetic products such aslip balms and other personal care products.

In general, over the counter (OTC) product and oral hygiene productgenerally refer to product for household and/or personal use which maybe sold without a prescription and/or without a visit to a medicalprofessional. Examples of the OTC products include, but are not limitedto Vitamins and dietary supplements; Topical analgesics and/oranaesthetic; oral analgesics (e.g., acetaminophen, ibuprofen, andaspirin); Cough, cold and allergy remedies; Antihistamines and/orallergy remedies (e.g., loratadine, diphenhydramine, fexofenadine, andcetirizine); digestive remedies (e.g., omeprazole, loperamide, calciumcarbonate, and psyllium seed husks) and combinations thereof. Vitaminsand dietary supplements include, but are not limited to vitamins,dietary supplements, tonics/bottled nutritive drinks, child-specificvitamins, dietary supplements, any other products of or relating to orproviding nutrition, and combinations thereof. Topical analgesics and/oranaesthetic include any topical creams/ointments/gels used to alleviatesuperficial or deep-seated aches and pains, e.g. muscle pain; teethinggel; patches with analgesic ingredient; and combinations thereof. Cough,cold and allergy remedies include, but are not limited to decongestants,cough remedies, pharyngeal preparations, medicated confectionery (e.g.,any candy confectionery product in any oral format that contains addedmenthol, pectin, vitamins, herbs and/or plant extracts, includingproducts for one or more of the following uses: nasal cooling, coughsuppression, breath freshening, sinus relief, dry mouth relief, mouthmoistening, throat soothing, and immunity boosting; the confectionerymay be in the form of, e.g., cough drops and throat drops),antihistamines and child-specific cough, cold and allergy remedies; andcombination products. Antihistamines and/or allergy remedies include,but are not limited to any systemic treatments for hay fever, nasalallergies, insect bites and stings. Examples of oral hygiene productsinclude, but are not limited to mouth cleaning strips, toothpaste,toothbrushes, mouthwashes/dental rinses, denture care, mouth freshenersat-home teeth whiteners and dental floss.

In another embodiment, the compounds of the present invention can beadded to food or beverage products or formulations. Examples of food andbeverage products or formulations include, but are not limited tocoatings, frostings, or glazes for comestible products or any entityincluded in the Soup category, the Dried Processed Food category, theBeverage category, the Ready Meal category, the Canned or Preserved Foodcategory, the Frozen Processed Food category, the Chilled Processed Foodcategory, the Snack Food category, the Baked Goods category, theConfectionary category, the Dairy Product category, the Ice Creamcategory, the Meal Replacement category, the Pasta and Noodle category,and the Sauces, Dressings, Condiments category, the Baby Food category,and/or the Spreads category.

In general, the Soup category refers to canned/preserved, dehydrated,instant, chilled, UHT and frozen soup. For the purpose of thisdefinition soup(s) means a food prepared from meat, poultry, fish,vegetables, grains, fruit and other ingredients, cooked in a liquidwhich may include visible pieces of some or all of these ingredients. Itmay be clear (as a broth) or thick (as a chowder), smooth, pureed orchunky, ready to serve, semi condensed or condensed and may be servedhot or cold, as a first course or as the main course of a meal or as abetween meal snack (sipped like a beverage). Soup may be used as aningredient for preparing other meal components and may range from broths(consomme) to sauces (cream or cheese based soups).

“Dehydrated and Culinary Food Category” usually means: (i) Cooking aidproducts such as: powders, granules, pastes, concentrated liquidproducts, including concentrated bouillon, bouillon and bouillon likeproducts in pressed cubes, tablets or powder or granulated form, whichare sold separately as a finished product or as an ingredient within aproduct, sauces and recipe mixes (regardless of technology); (ii) Mealsolutions products such as: dehydrated and freeze dried soups, includingdehydrated soup mixes, dehydrated instant soups, dehydrated ready tocook soups, dehydrated or ambient preparations of ready made dishes,meals and single serve entrees including pasta, potato and rice dishes;and (iii) Meal embellishment products such as: condiments, marinades,salad dressings, salad toppings, dips, breading, batter mixes, shelfstable spreads, barbecue sauces, liquid recipe mixes, concentrates,sauces or sauce mixes, including recipe mixes for salad, sold as afinished product or as an ingredient within a product, whetherdehydrated, liquid or frozen.

The Beverage category usually means beverages, beverage mixes andconcentrates, including but not limited to, carbonated and noncarbonated beverages, alcoholic and non alcoholic beverages, ready todrink beverages, liquid concentrate formulations for preparing beveragessuch as sodas, and dry powdered beverage precursor mixes. The Beveragecategory also include the alcoholic drinks, the soft drinks, sportsdrinks, isotonic beverages, and hot drinks. The alcoholic drinksinclude, but are not limited to beer, cider/perry, FABs, wine,absinthe-containing drinks, and spirits. The soft drinks include, butare not limited to carbonates, such a as colas and non-cola carbonates;fruit juice, such as juice, nectars, juice drinks and fruit flavoureddrinks; bottled water, which includes sparkling water, spring water andpurified/table water; functional drinks, which can be carbonated orstill and include sport, energy or elixir drinks; concentrates, such asliquid and powder concentrates in ready to drink measure. The hot drinksinclude, but are not limited to coffee, such as fresh (e.g., brewed),instant, combined coffee, liquid, ready-to-drink, soluble and dry coffeebeverages, coffee beverage mixes and concentrates (syrups, pure,formulated, or in powder form; example of a “powder form” is a productcomprising coffee, sweetener, and whitener all in powder form); tea,such as black, green, white, oolong, and flavored tea; and other hotdrinks including flavour-, malt- or plant-based powders, granules,blocks or tablets mixed with milk or water.

The Snack Food category generally refers to any food that can be a lightinformal meal including, but not limited to Sweet and savory snacks andsnack bars. Examples of snack food include, but are not limited to fruitsnacks, chips/crisps, extruded snacks, tortilla/corn chips, popcorn,pretzels, nuts and other sweet and savory snacks. Examples of snack barsinclude, but are not limited to granola/muesli bars, breakfast bars,energy bars, fruit bars and other snack bars.

The Baked Goods category generally refers to any edible product theprocess of preparing which involves exposure to heat or excessivesunlight. Examples of baked goods include, but are not limited to bread,buns, cookies, muffins, cereal, toaster pastries, pastries, waffles,tortillas, biscuits, pies, bagels, tarts, quiches, cake, any bakedfoods, and any combination thereof.

The Ice Cream category generally refers to frozen dessert containingcream and sugar and flavoring. Examples of ice cream include, but arenot limited to: impulse ice cream; take-home ice cream; frozen yoghurtand artisanal ice cream; soy, oat, bean (e.g., red bean and mung bean),and rice-based ice creams.

The Confectionary category generally refers to edible product that issweet to the taste. Examples of confectionary include, but are notlimited to candies, gelatins, chocolate confectionery, sugarconfectionery, gum, and the likes and any combination products.

The Meal Replacement category generally refers to any food intended toreplace the normal meals, particularly for people having health orfitness concerns. Examples of meal replacement include, but are notlimited to slimming products and convalescence products.

The Ready Meal category generally refers to any food that can be servedas meal without extensive preparation or processing. The read mealinclude products that have had recipe “skills” added to them by themanufacturer, resulting in a high degree of readiness, completion andconvenience. Examples of ready meal include, but are not limited tocanned/preserved, frozen, dried, chilled ready meals; dinner mixes;frozen pizza; chilled pizza; and prepared salads.

The Pasta and Noodle category includes any pastas and/or noodlesincluding, but not limited to canned, dried and chilled/fresh pasta; andplain, instant, chilled, frozen and snack noodles.

The Canned/Preserved Food category includes, but is not limited tocanned/preserved meat and meat products, fish/seafood, vegetables,tomatoes, beans, fruit, ready meals, soup, pasta, and othercanned/preserved foods.

The Frozen Processed Food category includes, but is not limited tofrozen processed red meat, processed poultry, processed fish/seafood,processed vegetables, meat substitutes, processed potatoes, bakeryproducts, desserts, ready meals, pizza, soup, noodles, and other frozenfood.

The Dried Processed Food category includes, but is not limited to rice,dessert mixes, dried ready meals, dehydrated soup, instant soup, driedpasta, plain noodles, and instant noodles.

The Chill Processed Food category includes, but is not limited tochilled processed meats, processed fish/seafood products, lunch kits,fresh cut fruits, ready meals, pizza, prepared salads, soup, fresh pastaand noodles.

The Sauces, Dressings and Condiments category includes, but is notlimited to tomato pastes and purees, bouillon/stock cubes, herbs andspices, monosodium glutamate (MSG), table sauces, soy based sauces,pasta sauces, wet/cooking sauces, dry sauces/powder mixes, ketchup,mayonnaise, mustard, salad dressings, vinaigrettes, dips, pickledproducts, and other sauces, dressings and condiments.

The Baby Food category includes, but is note limited to milk- orsoybean-based formula; and prepared, dried and other baby food.

The Spreads category includes, but is not limited to jams and preserves,honey, chocolate spreads, nut based spreads, and yeast based spreads.

The Dairy Product category generally refers to edible product producedfrom mammal's milk. Examples of dairy product include, but are notlimited to drinking milk products, cheese, yoghurt and sour milk drinks,and other dairy products.

Additional examples for comestible composition, particularly food andbeverage products or formulations, are provided as follows. Exemplarycomestible compositions include one or more confectioneries, chocolateconfectionery, tablets, countlines, bagged selflines/softlines, boxedassortments, standard boxed assortments, twist wrapped miniatures,seasonal chocolate, chocolate with toys, alfajores, other chocolateconfectionery, mints, standard mints, power mints, boiled sweets,pastilles, gums, jellies and chews, toffees, caramels and nougat,medicated confectionery, lollipops, liquorice, other sugarconfectionery, gum, chewing gum, sugarized gum, sugar free gum,functional gum, bubble gum, bread, packaged/industrial bread,unpackaged/artisanal bread, pastries, cakes, packaged/industrial cakes,unpackaged/artisanal cakes, cookies, chocolate coated biscuits, sandwichbiscuits, filled biscuits, savory biscuits and crackers, breadsubstitutes, breakfast cereals, rte cereals, family breakfast cereals,flakes, muesli, other cereals, children's breakfast cereals, hotcereals, ice cream, impulse ice cream, single portion dairy ice cream,single portion water ice cream, multi pack dairy ice cream, multi packwater ice cream, take home ice cream, take home dairy ice cream, icecream desserts, bulk ice cream, take home water ice cream, frozenyoghurt, artisanal ice cream, dairy products, milk, fresh/pasteurizedmilk, full fat fresh/pasteurized milk, semi skimmed fresh/pasteurizedmilk, long life/uht milk, full fat long life/uht milk, semi skimmed longlife/uht milk, fat free long life/uht milk, goat milk,condensed/evaporated milk, plain condensed/evaporated milk, flavored,functional and other condensed milk, flavored milk drinks, dairy onlyflavored milk drinks, flavored milk drinks with fruit juice, soy milk,sour milk drinks, fermented dairy drinks, coffee whiteners (e.g., dairyand non-dairy based creamers or whiteners for coffee beverages), powdermilk, flavored powder milk drinks, cream, cheese, processed cheese,spreadable processed cheese, unspreadable processed cheese, unprocessedcheese, spreadable unprocessed cheese, hard cheese, packaged hardcheese, unpackaged hard cheese, yoghurt, plain/natural yoghurt, flavoredyoghurt, fruited yoghurt, probiotic yoghurt, drinking yoghurt, regulardrinking yoghurt, probiotic drinking yoghurt, chilled and shelf stabledesserts, dairy based desserts, soy based desserts, chilled snacks,fromage frais and quark, plain fromage frais and quark, flavored fromagefrais and quark, savory fromage frais and quark, sweet and savorysnacks, fruit snacks, chips/crisps, extruded snacks, tortilla/cornchips, popcorn, pretzels, nuts, other sweet and savory snacks, snackbars, granola bars, breakfast bars, energy bars, fruit bars, other snackbars, meal replacement products, slimming products, convalescencedrinks, ready meals, canned ready meals, frozen ready meals, dried readymeals, chilled ready meals, dinner mixes, frozen pizza, chilled pizza,soup, canned soup, dehydrated soup, instant soup, chilled soup, hotsoup, frozen soup, pasta, canned pasta, dried pasta, chilled/freshpasta, noodles, plain noodles, instant noodles, cups/bowl instantnoodles, pouch instant noodles, chilled noodles, snack noodles, cannedfood, canned meat and meat products, canned fish/seafood, cannedvegetables, canned tomatoes, canned beans, canned fruit, canned readymeals, canned soup, canned pasta, other canned foods, frozen food,frozen processed red meat, frozen processed poultry, frozen processedfish/seafood, frozen processed vegetables, frozen meat substitutes,frozen potatoes, oven baked potato chips, other oven baked potatoproducts, non oven frozen potatoes, frozen bakery products, frozendesserts, frozen ready meals, frozen pizza, frozen soup, frozen noodles,other frozen food, dried food, dessert mixes, dried ready meals,dehydrated soup, instant soup, dried pasta, plain noodles, instantnoodles, cups/bowl instant noodles, pouch instant noodles, chilled food,chilled processed meats, chilled fish/seafood products, chilledprocessed fish, chilled coated fish, chilled smoked fish, chilled lunchkit, chilled ready meals, chilled pizza, chilled soup, chilled/freshpasta, chilled noodles, oils and fats, olive oil, vegetable and seedoil, cooking fats, butter, margarine, spreadable oils and fats,functional spreadable oils and fats, sauces, dressings and condiments,tomato pastes and purees, bouillon/stock cubes, stock cubes, gravygranules, liquid stocks and fonds, herbs and spices, fermented sauces,soy based sauces, pasta sauces, wet sauces, dry sauces/powder mixes,ketchup, mayonnaise, regular mayonnaise, mustard, salad dressings,regular salad dressings, low fat salad dressings, vinaigrettes, dips,pickled products, other sauces, dressings and condiments, baby food,milk formula, standard milk formula, follow on milk formula, toddlermilk formula, hypoallergenic milk formula, prepared baby food, driedbaby food, other baby food, spreads, jams and preserves, honey,chocolate spreads, nut based spreads, and yeast based spreads. Exemplarycomestible compositions also include confectioneries, bakery products,ice creams, dairy products, sweet and savory snacks, snack bars, mealreplacement products, ready meals, soups, pastas, noodles, canned foods,frozen foods, dried foods, chilled foods, oils and fats, baby foods, orspreads or a mixture thereof. Exemplary comestible compositions alsoinclude breakfast cereals, sweet beverages or solid or liquidconcentrate compositions for preparing beverages. Exemplary comestiblecompositions also include coffee flavored food (e.g., coffee flavoredice cream).

In some embodiments, the compounds of the present invention can be addedto a comestible composition, particularly food and beverage products orformulations, including, without limitation, cereal products, riceproducts, tapioca products, sago products, baker's products, biscuitproducts, pastry products, bread products, confectionery products,desert products, gums, chewing gums, chocolates, ices, honey products,treacle products, yeast products (e.g., yeast extract, tortula yeast,and brewers yeast and other savory enhancer ingredients), baking-powder,salt and spice products, savory products, mustard products, vinegarproducts, sauces (condiments), tobacco products, cigars, cigarettes,processed foods, cooked fruits and vegetable products, meat and meatproducts, jellies, jams, fruit sauces, egg products, milk and dairyproducts, yoghurts, cheese products, butter and butter substituteproducts, milk substitute products, soy products, edible oils and fatproducts, medicaments, beverages, carbonated beverages, alcoholicdrinks, beers, soft drinks, mineral and aerated waters and othernon-alcoholic drinks (e.g., beverages functioning as dietary supplementsand/or an added source of protein), fruit drinks, fruit juices, coffee,artificial coffee, tea, cocoa, including forms requiring reconstitution,food extracts, plant extracts, meat extracts, condiments, sweeteners,nutraceuticals, gelatins, pharmaceutical and non-pharmaceutical gums,tablets, lozenges, drops, emulsions, elixirs, syrups and otherpreparations for making beverages, and combinations thereof. Othercomestible compositions include, without limitation, any comestiblecomposition comprising caffeine and other methylxanthines (e.g.,theophylline; theobromine; and nicotine, which can be contained in, forexample, chewing gum to help to quit smoking).

In other embodiments, the compounds of the present invention can beadded to a comestible composition, particularly food and beverageproducts or formulations, including, without limitation, water-basedcompositions including, without limitation, the following: aqueousdrink, enhanced/slightly sweetened water drink, carbonated beverage,non-carbonated beverage, soft drink, non-alcoholic drink, alcoholicdrink, fruit drink, juice, fruit juice, vegetable juice, coffee (iced orhot), tea, black tea (iced or hot and extracts thereof), green tea (icedor hot and extracts thereof), oolong tea (iced or hot), herbal tea (icedor hot), mate tea (iced or hot), cocoa (water-based), cocoa(milk-based), cocoa (soy-based), tea-based drink, coffee-based drink,cocoa-based drink, syrup, frozen fruit, frozen fruit juice, water-basedice, dairy ice, fruit ice, sorbet, and beverages formed from botanicalmaterials (whole or ground) by brewing, soaking or otherwise extracting,and beverages formed by dissolving instant powders or concentrates(coffee beans, ground coffee, instant coffee, cocoa beans, cocoa powder,instant cocoa, tea leaves, instant tea powder), and the above-mentionedconcentrates.

In still other embodiments, the compounds of the present invention canbe added to a comestible composition, particularly food and beverageproducts or formulations, including, without limitation, solid drycompositions including, without limitation, cereals, baked foodproducts, biscuits, bread, breakfast cereal, cereal bar, energybars/nutritional bars, granola, cakes, cookies, crackers, donuts,muffins, pastries, confectioneries, chewing gum, chocolate, fondant,hard candy, marshmallow, pressed tablets, snack foods, and botanicalmaterials (whole or ground), and instant powders for reconstitution asmentioned herein above.

In yet other embodiments, the compounds of the present invention can beadded to a comestible composition, particularly food and beverageproducts or formulations, including, without limitation, dairy products,dairy-derived products and dairy-alternative products, including,without limitation, milk, fluid milk, cultured milk product, culturedand noncultured dairy-based drinks, cultured milk product cultured withlactobacillus, yoghurt, yoghurt-based beverage, smoothy, lassi, milkshake, acidified milk, acidified milk beverage, butter milk, kefir,milk-based beverage, milk/juice blend, fermented milk beverage,icecream, dessert, frozen yoghurt, soy milk, rice milk, soy drink, ricemilk drink. Milk includes, but is not limited to, whole milk, skim milk,condensed milk, evaporated milk, reduced fat milk, low fat milk, nonfatmilk, and milk solids (which may be fat or nonfat).

In other embodiments, the compounds of the present invention can beadded to a comestible composition, particularly food and beverageproducts or formulations, including, without limitation, compositionscomprising one or more ingredient selected from the group consisting ofone or more vitamins provided as an additive; one or more B-vitaminscholine, inositol, riboflavin, thiamin) provided as an additive; one ormore amino acids; coffee or an extract thereof (Coffea spec.): cocoa oran extract thereof (Theobroma spec.); guarana or an extract thereof(Paullinia cupana, P. crysan, or P. sorbilis); black tea or an extractthereof, green tea or an extract thereof, yerba mate or an extractthereof (Camellia spec. extracts), Yaupon/Cassina extracts (Ilexvomitoria); taurine; ginseng or an extract thereof; kola or an extractthereof (Cola acuminata); carob or an extract thereof (Ceratoniasiliqua); maltodextrin; inositol; carnitine; creatine; glucuronolactone;ginkgo biloba extract; golden seal or an extract thereof; and dandelionor an extract thereof.

In other embodiments, the compounds of the present invention can beadded to a comestible composition, particularly food and beverageproducts or formulations, including, without limitation, compositionscomprising trilobatin extracted from a botanical source, optionallyselected from parts or leaves of Lithocarpus polystachyus (Chinese sweettea) and parts or leaves of an apple species, said apple speciesoptionally being selected from Malus trilobata.

In other embodiments, the compounds of the present invention can beadded to a comestible composition, particularly food and beverageproducts or formulations, including, without limitation, compositionscomprising vegetable and/or non-vegetable proteins. As used herein, theterm “non-vegetable protein(s)” means any protein(s), with the exceptionof vegetable proteins. Examples of non-vegetable proteins include, butare not limited to proteins derived from milk (e.g., whey proteins,isolates and other dairy hydrolysates such as milk casein hydrolysates).As used herein, the term “vegetable proteins” means any plant andvegetable protein(s) including, without limitation, proteins from grains(e.g., wheat, corn, barley, oats, rye, millet, and buckwheat); proteinsfrom nuts (e.g., walnuts, cashews, almonds, pecans); soy protein(s)(hydrolyzed and unhydrolyzed); proteins from seeds (e.g., sunflower,pumpkin, hemp, and flax); proteins from legumes (e.g., beans, lentils,and garbanzos (chickpeas)); and proteins from rice and pea isolates.

In other embodiments, the compounds of the present invention can beadded to a comestible composition, particularly food and beverageproducts or formulations, including, without limitation, compositionscomprising menthol, goji berry juice (or an extract thereof),pomegranate extract, grapeseed extract, rhodiola (or an extractthereof), black cohosh (or an extract thereof; also known as Actaearacemosa and Cimicifuga racemosa). artificial sweeteners (e.g.aspartame, saccharin, sucralose, and acesulfame-K), cooling agents(e.g., menthane carboxamide), spirulina, yerba mate (or extractsthereof), spirulina (or extracts thereof), chicory (or extractsthereof), and natural sweeteners (e.g. Luo Han Guo, monatin, andstevia), including sweeteners comprising stevioside, steviol glycosides,rebaudioside A, rebaudioside C, and/or dulcoside A.

In other embodiments, the compounds of the present invention can beadded to a comestible composition, particularly food and beverageproducts or formulations, including, without limitation, hot or coldchocolate and/or malt-based flavored drinks including, withoutlimitation, in powdered, granulated or in a block/tablet form, or inready to drink form.

In other embodiments, the compounds of the present invention can beadded to a comestible composition, particularly food and beverageproducts or formulations, including, without limitation, compositionscomprising isoflavones, citrus oils (e.g., orange extract, lemonextract, lime extract, and combinations thereof) savory ingredients(e.g., hydrolyzed vegetable proteins), potassium chloride, variousleavening agents (e.g., beer, buttermilk, ginger beer, kefir, sourdoughstarter, yeast, yogurt, baking powder, baking soda (sodium bicarbonate),monocalcium phosphate, sodium aluminum phosphate, sodium acidpyrophosphate, other phosphates, ammonium bicarbonate (hartshorn, hornsalt, bakers ammonia), potassium bicarbonate (potash), potassiumbitartrate (cream of tartar), potassium carbonate (pearlash), andhydrogen peroxide) or chocolate liquor.

Typically an amount sufficient to alleviate or reduce the bitter tasteassociated with a composition, e.g., an ingestible composition, is addedto the composition to alleviate or reduce the bitter taste associatedwith the composition as compared to compositions that are preparedwithout the compounds of the present invention, as judged by humanbeings or animals. Or, in the case of formulations testing, as judged bya majority of a panel of, e.g., eight human taste testers, viaprocedures commonly known in the field.

The concentration of the compounds of the present invention effective toalleviate or reduce the bitter taste associated with a composition willof course depend on many variables, including the specific type ofcomestible composition and its various other ingredients, the naturalgenetic variability and individual preferences and health conditions ofvarious human beings tasting the compositions, and the subjective effectof the particular compound on the taste of such chemosensory compounds.In some embodiments, concentration of the compounds of the presentinvention effective to alleviate or reduce the bitter taste associatedwith a composition is from about 0.001 ppm to about 100 ppm, e.g., fromabout 0.1 ppm to about 100 ppm, from about 1 ppm to about 25 ppm, fromabout 1 ppm to about 10 ppm, from about 0.1 ppm to about 10 ppm, fromabout 0.01 ppm to about 30 ppm, from about 0.05 ppm to about 10 ppm,from about 0.01 ppm to about 5 ppm, from about 0.02 ppm to about 2 ppm,or from about 0.01 ppm to about 1 ppm.

It is contemplated that in some embodiments of the present invention, amixture of one or more compounds of the present invention will be usedto alleviate or reduce the bitter taste associated with a composition.The concentration of the one or more compounds may be the same or theconcentration of each compound may be different.

Prior to further describing the invention, the following definitions areprovided.

The term “T2R” family includes polymorphic variants, alleles, mutants,and homologs that: (1) have about 30-40% amino acid sequence identity,more specifically about 40, 50, 60, 70, 75, 80, 85, 90, 95, 96, 97, 98,or 99% amino acid sequence identity to the T2Rs disclosed infra, and inthe Zuker (Id) (2001) and Adler (Id.) (2001) applications incorporated,by reference herein over a window of about 25 amino acids, optimally50-100 amino acids; (2) specifically bind to antibodies raised againstan immunogen comprising an amino acid sequence selected from the groupconsisting of the T2R sequences disclosed infra, and conservativelymodified variants thereof; (3) specifically hybridize (with a size of atleast about 100, optionally at least about 500-1000 nucleotides) understringent hybridization conditions to a sequence selected from the groupconsisting of the T2R DNA sequences disclosed infra, and conservativelymodified variants thereof; (4) comprise a sequence at least about 40%identical to an amino acid sequence selected from the group consistingof the T2R amino acid sequences disclosed infra or (5) are amplified byprimers that specifically hybridize under stringent hybridizationconditions to the described T2R sequences.

In particular, these “T2R's” include taste receptor GPCRs referred toherein as hT2R8 and hT2R14 having the nucleic acid sequences and aminoacid sequences provided in this application, and variants, alleles,mutants, orthologs and chimeras thereof which specifically bind tobitter ligands which are identified herein and other structurallyrelated compounds and bitter compounds.

While T2R genes exhibit substantial sequence divergence at both theprotein and DNA level, all T2Rs isolated to date have been found tocontain certain consensus sequences in particular regions that areidentical or which possess or at least 70-75% sequence identity to theT2R consensus sequence identified previously in the Adler et al (WO01/77676 A1 (2001) and Zuker et al. WO 01/18050 A2, both incorporated byreference in their entirety herein.

Topologically, certain chemosensory GPCRs have an “N-terminal domain;”“extracellular domains,” a “transmembrane domain” comprising seventransmembrane regions, and corresponding cytoplasmic and extracellularloops, “cytoplasmic regions,” and a “C-terminal region” (see, e.g., Hoonet al, Cell, 96:541-51 (1999); Buck & Axel, Cell, 65:175-87 (1991)).These regions can be structurally identified using methods known tothose of skill in the art, such as sequence analysis programs thatidentify hydrophobic and hydrophilic domains (see, e.g., Stryer,Biochemistry, (3rd ed. 1988); see also any of a number of Internet basedsequence analysis programs, such as those found atdot.imgen.bcm.tmc.edu). These regions are useful for making chimericproteins and for in vitro assays of the invention, e.g., ligand bindingassays. For example chimeric T2Rs can be made by combining theextracellular region of one T2R and the transmembrane region of anotherT2R of the same or different species.

“Extracellular domains” therefore refers to the domains of T2Rpolypeptides that protrude from the cellular membrane and are exposed tothe extracellular face of the cell. Such regions would-include the“N-terminal domain” that is exposed to the extracellular face of thecell, as well as the extracellular loops of the transmembrane domainthat are exposed to the extracellular face of the cell, i.e., theextracellular loops between transmembrane regions 2 and 3, transmembraneregions 4 and 5, and transmembrane regions 6 and 7. The “N-terminaldomain” starts at the N-terminus and extends to a region close to thestart of the transmembrane region. These extracellular regions areuseful for in vitro ligand binding assays, both soluble and solid phase.In addition, transmembrane regions, described below, can also beinvolved in ligand binding, either in combination with the extracellularregion or alone, and are therefore also useful for in vitro ligandbinding assays.

“T2R Expressing Cell” herein encompasses recombinant cells which expressa human T2R sequence according to the invention as well as endogenousT2R expressing cells. Such cells are comprised in the lingual andgastrointestinal system and include cells in the oral cavity such astaste buds expressed on the tongue as well as cells in thegastrointestinal system and associated organs such as brush cells in thegastrointestinal tract, enteroendocrine cells such as STC-1 cells. Thesecells may also express a G protein such as gustducin, transducin,G_(α15) or G_(α16). Cells which express specific T2Rs can be identifiedand isolated by known methods such as by FACS cell separation and/ormagnetic bead cell isolation procedures.

“Transmembrane domain,” which comprises the seven transmembrane“regions,” refers to the domain of T2R polypeptides that lies within theplasma membrane, and may also include the corresponding cytoplasmic(intracellular) and extracellular loops, also referred to astransmembrane “regions.” The seven transmembrane regions andextracellular and cytoplasmic loops can be identified using standardmethods, as described in Kyte & Doolittle, J. Mol. Biol., 157:105-32(1982)), or in Stryer, supra.

“Cytoplasmic domains” refers to the domains of T2R proteins that facethe inside of the cell, e.g., the “C-terminal domain” and theintracellular loops of the transmembrane domain, e.g., the intracellularloops between transmembrane regions 1 and 2, transmembrane regions 3 and4, and transmembrane regions 5 and 6. “C-terminal domain” refers to theregion that spans from the end of the last transmembrane region to theC-terminus of the protein, and which is normally located within thecytoplasm.

The term “7-transmembrane receptor” means a polypeptide belonging to asuperfamily of transmembrane proteins that have seven regions that spanthe plasma membrane seven times (thus, the seven regions are called“transmembrane” or “TM” domains TM I to TM VII). The families ofolfactory and certain taste receptors each belong to this super-family.7-transmembrane receptor polypeptides have similar and characteristicprimary, secondary and tertiary structures, as discussed in furtherdetail below.

The term “ligand-binding region” refers to sequences derived from achemosensory or taste receptor that substantially incorporatestransmembrane domains II to VII (TM II to VII). The region may becapable of binding a ligand, and more particularly, a taste elicitingcompound.

The term “plasma membrane translocation domain” or simply “translocationdomain” means a polypeptide domain which when incorporated into theamino terminus of a polypeptide coding sequence, can with greatefficiency “chaperone” or “translocate” the hybrid (“fusion”) protein tothe cell plasma membrane. For example a particular “translocationdomain” initially derived from the amino terminus of the human rhodopsinreceptor polypeptide, a 7-transmembrane receptor can be used. Anothertranslocation domain has been derived from the bovine rhodopsin sequenceand is also useful for facilitating translocation. Rhodopsin derivedsequences are particularly efficient in translocating 7-transmembranefusion proteins to the plasma membrane.

“Functional equivalency” means the domain's ability and efficiency intranslocating newly translated proteins to the plasma membrane asefficiently as an exemplary translocation domain such as one derivedfrom rhodopsin under similar conditions; relative efficiencies can bemeasured (in quantitative terms) and compared, as described herein.Domains falling within the scope of the invention can be determined byroutine screening for their efficiency in translocating newlysynthesized polypeptides to the plasma membrane in a cell (mammalian,Xenopus, and the like) with the same efficiency as the twenty amino acidlong translocation domain SEQ ID NO:1.

The phrase “functional effects” in the context of assays for testingcompounds that modulate T2R family member mediated taste transductionincludes the determination of any parameter that is indirectly ordirectly under the influence of the receptor, e.g., functional, physicaland chemical effects. It includes ligand binding, changes in ion flux,membrane potential, current flow, transcription, G protein binding, GPCRphosphorylation or dephosphorylation, signal transduction,receptor-ligand interactions, second messenger concentrations (e.g.,cAMP, cGMP, IP3, or intracellular Ca²⁺), in vitro, in vivo, and ex vivoand also includes other physiologic effects such increases or decreasesof neurotransmitter or hormone release.

By “determining the functional effect” is meant assays for a compoundthat increases or decreases a parameter that is indirectly or directlyunder the influence of a T2R family member, e.g., functional, physicaland chemical effects. Such functional effects can be measured by anymeans known to those skilled in the art, e.g., changes in spectroscopiccharacteristics (e.g., fluorescence, absorbance, refractive index),hydrodynamic (e.g., shape), chromatographic, or solubility properties,patch clamping, voltage-sensitive dyes, whole cell currents,radioisotope efflux, inducible markers, oocyte T2R gene expression;tissue culture cell T2R expression; transcriptional activation of T2Rgenes; ligand binding assays; voltage, membrane potential andconductance changes; ion flux assays; changes in intracellular secondmessengers such as cAMP, cGMP, and inositol triphosphate (IP3); changesin intracellular calcium levels; neurotransmitter release, and the like.

“Inhibitors,” “activators,” and “modulators” of T2R proteins receptorsare used interchangeably to refer to inhibitory, activating, ormodulating molecules identified using in vitro and in vivo assays fortaste transduction, e.g., ligands, agonists, antagonists, and theirhomologs and mimetics. Inhibitors are compounds that, e.g., bind to,partially or totally block stimulation, decrease, prevent, delayactivation, inactivate, desensitize, or down regulate tastetransduction, e.g., antagonists. Activators are compounds that, e.g.,bind to, stimulate, increase, open, activate, facilitate, enhanceactivation, sensitize, or up regulate taste transduction, e.g.,agonists. Modulators include compounds that, e.g., alter the interactionof a receptor with extracellular proteins that bind activators orinhibitor (e.g., ebnerin and other members of the hydrophobic carrierfamily); G Proteins; kinases (e.g., homologs of rhodopsin kinase andbeta adrenergic receptor kinases that are involved in deactivation anddesensitization of a receptor); and arrestins, which also deactivate anddesensitize receptors. Modulators include genetically modified versionsof T2R family members, e.g., with altered activity, as well as naturallyoccurring and synthetic ligands, antagonists, agonists, small chemicalmolecules and the like.

Such assays for inhibitors and activators include, e.g., expressing T2Rfamily members in cells or cell membranes, applying putative modulatorcompounds in the presence or absence of compounds that modulate, e.g.,bitter compounds, and then determining the functional effects on tastetransduction, as described above. Samples or assays comprising T2Rfamily members that are treated with a potential activator, inhibitor,or modulator are compared to control samples without the inhibitor,activator, or modulator to examine the extent of modulation. Controlsamples (untreated with modulators) are assigned a relative T2R activityvalue of 100%. Inhibition of a T2R is achieved when the T2R activityvalue relative to the control is about 80%, optionally 50% or 25-0%.Activation of a T2R is achieved when the T2R activity value relative tothe control is 110%, optionally 150%, optionally 200-500%, or 1000-3000%higher.

The terms “purified,” “substantially purified,” and “isolated” as usedherein refer to the state of being free of other, dissimilar compoundswith which the compound of the invention is normally associated in itsnatural state. Preferably, “purified,” “substantially purified,” and“isolated” means that the composition comprises at least 0.5%, 1%, 5%,10%, or 20%, and most preferably at least 50% or 75% of the mass, byweight, of a given sample. In one preferred embodiment, these termsrefer to the compound of the invention comprising at least 95% of themass, by weight, of a given sample. As used herein, the terms“purified,” “substantially purified,” and “isolated”, when referring toa nucleic acid or protein, of nucleic acids or proteins, also refers toa state of purification or concentration different than that whichoccurs naturally in the mammalian, especially human, body. Any degree ofpurification or concentration greater than that which occurs naturallyin the mammalian, especially human, body, including (1) the purificationfrom other associated structures or compounds or (2) the associationwith structures or compounds to which it is not normally associated inthe mammalian, especially human, body, are within the meaning of“isolated.” The nucleic acid or protein or classes of nucleic acids orproteins, described herein, may be isolated, or otherwise associatedwith structures or compounds to which they are not normally associatedin nature, according to a variety of methods and processes known tothose of skill in the art.

As used herein, the term “isolated,” when referring to a nucleic acid orpolypeptide refers to a state of purification or concentration differentthan that which occurs naturally in the mammalian, especially human,body. Any degree of purification or concentration greater than thatwhich occurs naturally in the body, including (1) the purification fromother naturally-occurring associated structures or compounds, or (2) theassociation with structures or compounds to which it is not normallyassociated in the body are within the meaning of “isolated” as usedherein. The nucleic acids or polypeptides described herein may beisolated or otherwise associated with structures or compounds to whichthey are not normally associated in nature, according to a variety ofmethods and processed known to those of skill in the art.

As used herein, the terms “amplifying” and “amplification” refer to theuse of any suitable amplification methodology for generating ordetecting recombinant or naturally expressed nucleic acid, as describedin detail, below. For example, the invention provides methods andreagents (e.g., specific oligonucleotide primer pairs) for amplifying(e.g., by polymerase chain reaction, PCR) naturally expressed (e.g.,genomic or mRNA) or recombinant (e.g., cDNA) nucleic acids of theinvention (e.g., taste eliciting compound-binding sequences of theinvention) in vivo or in vitro.

The term “expression vector” refers to any recombinant expression systemfor the purpose of expressing a nucleic acid sequence of the inventionin vitro or in vivo, constitutively or inducibly, in any cell, includingprokaryotic, yeast, fungal, plant, insect or mammalian cell. The termincludes linear or circular expression systems. The term includesexpression systems that remain episomal or integrate into the host cellgenome. The expression systems can have the ability to self-replicate ornot, i.e., drive only transient expression in a cell. The term includesrecombinant expression “cassettes which contain only the minimumelements needed for transcription of the recombinant nucleic acid.

The term “library” means a preparation that is a mixture of differentnucleic acid or poly-peptide molecules, such as the library ofrecombinant generated sensory, particularly taste receptorligand-binding regions generated by amplification of nucleic acid withdegenerate primer pairs, or an isolated collection of vectors thatincorporate the amplified ligand-binding regions, or a mixture of cellseach randomly transfected with at least one vector encoding an tastereceptor.

The term “nucleic acid” or “nucleic acid sequence” refers to adeoxy-ribonucleotide or ribonucleotide oligonucleotide in either single-or double-stranded form. The term encompasses nucleic acids, i.e.,oligonucleotides, containing known analogs of natural nucleotides. Theterm also encompasses nucleic-acid-like structures with syntheticbackbones.

Unless otherwise indicated, a particular nucleic acid sequence alsoimplicitly encompasses conservatively modified variants thereof (e.g.,degenerate codon substitutions) and complementary sequences, as well asthe sequence explicitly indicated. Specifically, degenerate codonsubstitutions may be achieved by generating, e.g., sequences in whichthe third position of one or more selected codons is substituted withmixed-base and/or deoxyinosine residues (Batzer et al., Nucleic AcidRes., 19:5081 (1991); Ohtsuka et al., J. Biol. Chem., 260:2605-08(1985); Rossolini et al., Mol. Cell. Probes, 8:91-98 (1994)). The termnucleic acid is used interchangeably with gene, cDNA, mRNA,oligonucleotide, and polynucleotide.

The terms “polypeptide,” “peptide” and “protein” are usedinterchangeably herein to refer to a polymer of amino acid residues. Theterms apply to amino acid polymers in which one or more amino acidresidue is an artificial chemical mimetic of a corresponding naturallyoccurring amino acid, as well as to naturally occurring amino acidpolymers and non-naturally occurring amino acid polymer.

As used herein, “alkyl” as well as other groups having the prefix “alk”such as, for example, alkoxy, alkanoyl, alkenyl, alkynyl and the like,means carbon chains which may be linear or branched or combinationsthereof. Examples of alkyl groups include methyl, ethyl, propyl,isopropyl, butyl, sec- and tert-butyl, pentyl, hexyl, heptyl and thelike. Preferred alkyl groups have 1-4 carbons. “Alkenyl” and other liketerms include carbon chains containing at least one unsaturatedcarbon-carbon bond. “Alkynyl” and other like terms include carbon chainscontaining at least one carbon-carbon triple bond.

The term “cycloalkyl” means carbocycles containing no heteroatoms, andincludes mono-, bi- and tricyclic saturated carbocycles, as well asfused ring systems. Examples of cycloalkyl include cyclopropyl,cyclobutyl, cyclopentyl, cyclohexyl, decahydronaphthalene, adamantane,indanyl, indenyl, fluorenyl, 1,2,3,4-tetrahydronaphthalene and the like.

The term “aryl” means an aromatic substituent that is a single ring ormultiple rings fused together. Exemplary aryl groups include, withoutlimitation, phenyl, naphthyl, anthracenyl, pyridinyl, pyrazinyl,pyrimidinyl, triazinyl, thiophenyl, furanyl, pyrrolyl, oxazolyl,pyrazolyl, imidazolyl, triazyolyl, and tetrazolyl groups. Aryl groupsthat contain one or more heteroatoms (e.g., pyridinyl) are oftenreferred to as “heteroaryl groups.” When formed of multiple rings, atleast one of the constituent rings is aromatic. In some embodiments, atleast one of the multiple rings comprise a heteroatom, thereby formingheteroatom-containing aryl groups. Heteroatom-containing aryl groupsinclude, without limitation, benzoxazolyl, benzimidazolyl, quinoxalinyl,benzofuranyl, and 1H-benzo[d][1,2,3]triazolyl groups.Heteroatom-containing aryl groups also include, without limitation,2,3-dihydrobenzo[b][1,4]dioxinyl and benzo[d][1,3]dioxolyl groups.Heteroatom-containing aryl groups also include aromatic rings fused to aheterocyclic ring comprising at least one heteroatom and at least onecarbonyl group. Such groups include, without limitation, dioxotetrahydroquinoxalinyl and dioxo tetrahydroquinazolinyl groups.

The term “acyl” means a R—C(O)— group.

The term “arylalkoxy” means an aryl group bonded to an alkoxy group.

The term “arylamidoalkyl” means an aryl-C(O)NR-alkyl oraryl-NRC(O)-alkyl.

The term “arylalkylamidoalkyl” means an aryl-alkyl-C(O)NR-alkyl oraryl-alkyl-NRC(O)-alkyl, wherein R is anyl suitable group listed below.

The term “arylalkyl” refers to an aryl group bonded to an alkyl group.

The term “halogen” or “halo” refers to chlorine, bromine, fluorine oriodine.

The term “leaving group” refers to a functional group or atom which canbe displaced by another functional group or atom in a substitutionreaction, such as a nucleophilic substitution reaction. By way ofexample, representative leaving groups include chloro, bromo and iodogroups; sulfonic ester groups, such as mesylate, tosylate, brosylate,nosylate and the like; and acyloxy groups, such as acetoxy,trifluoroacetoxy and the like.

The term “haloalkyl” means an alkyl group having one or more halogenatoms (e.g., CF₃).

The term “heteroalkyl” refers to an alkyl moiety which comprises aheteroatom such as N, O, P, B, S, or Si. The heteroatom may be connectedto the rest of the heteroalkyl moiety by a saturated or unsaturatedbond. Thus, an alkyl substituted with a group, such as heterocycloalkyl,substituted heterocycloalkyl, heteroaryl, substituted heteroaryl,alkoxy, aryloxy, boryl, phosphino, amino, silyl, thio, or seleno, iswithin the scope of the term heteroalkyl. Examples of heteroalkylsinclude, but are not limited to, cyano, benzoyl, 2-pyridyl and 2-furyl.

The term “heteroarylalkyl” means a heteroaryl group to which an alkylgroup is attached.

The term “heterocycle” means a monocyclic or polycyclic ring comprisingcarbon and hydrogen atoms, optionally having 1 or 2 multiple bonds, andthe ring atoms contain at least one heteroatom, specifically 1 to 4heteroatoms, independently selected from nitrogen, oxygen, and sulfur.Heterocycle ring structures include, but are not limited to, mono-, bi-,and tri-cyclic compounds. Specific heterocycles are monocyclic orbicyclic. Representative heterocycles include cyclic ureas, morpholinyl,pyrrolidinonyl, pyrrolidinyl, piperidinyl, piperazinyl, hydantoinyl,valerolactamyl, oxiranyl, oxetanyl, tetrahydrofuranyl,tetrahydropyranyl, tetrahydropyridinyl, tetrahydroprimidinyl,tetrahydrothiophenyl, tetrahydrothiopyranyl, tetrazolyl, and urazolyl. Aheterocyclic ring may be unsubstituted or substituted. Preferredheterocycles are 5- and 6-membered heterocycles, particularlyhydantoinyl and urazolyl.

The term “heterocycloalkyl” or “cycloheteroalkyl” refers to a cycloalkylgroup in which at least one of the carbon atoms in the ring is replacedby a heteroatom (e.g., O, S or N). In some embodiments, theheterocycloalkyl group may have one or more unsaturated bonds. Inanother embodiment, the heterocycloalkyl group is fused with anothercycloalkyl, heterocycloalkyl, aryl or heteroaryl group.

The term “heterocycloalkylalkyl” means a heterocycloalkyl group to whichthe an alkyl group is attached.

The term “substituted” specifically envisions and allows for one or moresubstitutions that are common in the art. However, it is generallyunderstood by those skilled in the art that the substituents should beselected so as to not adversely affect the useful characteristics of thecompound or adversely interfere with its function. Suitable substituentsmay include, for example, halo groups, perfluoroalkyl groups,perfluoroalkoxy groups, alkyl groups, alkenyl groups, alkynyl groups,hydroxy groups, oxo groups, mercapto groups, alkylthio groups, alkoxygroups, aryl or heteroaryl groups, aryloxy or heteroaryloxy groups,aralkyl or heteroaralkyl groups, aralkoxy or heteroaralkoxy groups,amino groups, alkyl- and dialkylamino groups, carbamoyl groups,alkylcarbonyl groups, carboxyl groups, alkoxycarbonyl groups, alkylaminocarbonyl groups, dialkylamino carbonyl groups, arylcarbonyl groups,aryloxycarbonyl groups, alkylsulfonyl groups, arylsulfonyl groups,cycloalkyl groups, cyano groups, C₁-C₆ alkylthio groups, arylthiogroups, nitro groups, keto groups, acyl groups, boronate or boronylgroups, phosphate or phosphoryl groups, sulfamyl groups, sulfonylgroups, sulfinyl groups, and combinations thereof. In the case ofsubstituted combinations, such as “substituted arylalkyl,” either thearyl or the alkyl group may be substituted, or both the aryl and thealkyl groups may be substituted with one or more substituents.Additionally, in some cases, suitable substituents may combine to formone or more rings as known to those of skill in the art.

Compounds described herein contain one or more double bonds and may thusgive rise to cis/trans isomers as well as other conformational isomers.The present invention includes all such possible isomers as well asmixtures of such isomers.

Compounds described herein, and particularly the substituents describedabove, may contain one or more asymmetric centers and may thus give riseto diastereomers and optical isomers. The present invention includes allsuch possible diastereomers as well as their racemic mixtures, theirsubstantially pure resolved enantiomers, all possible geometric isomers,and acceptable salts thereof. Further, mixtures of stereoisomers as wellas isolated specific stereoisomers are also included. During the courseof the synthetic procedures used to prepare such compounds, or in usingracemization or epimerization procedures known to those skilled in theart, the products of such procedures can be a mixture of stereoisomers.

As used herein, the term “salts” and “pharmaceutically acceptable salts”refer to derivatives of the disclosed compounds wherein the parentcompound is modified by making acid or base salts thereof. Examples ofpharmaceutically acceptable salts include, but are not limited to,mineral or organic acid salts of basic groups such as amines; and alkalior organic salts of acidic groups such as carboxylic acids.Pharmaceutically acceptable salts include the conventional non-toxicsalts or the quaternary ammonium salts of the parent compound formed,for example, from non-toxic inorganic or organic acids. For example,such conventional non-toxic salts include those derived from inorganicacids such as hydrochloric, hydrobromic, sulfuric, sulfamic, phosphoric,and nitric; and the salts prepared from organic acids such as acetic,propionic, succinic, glycolic, stearic, lactic, malic, tartaric, citric,ascorbic, pamoic, maleic, hydroxymaleic, phenylacetic, glutamic,benzoic, salicylic, sulfanilic, 2-acetoxybenzoic, fumaric,toluenesulfonic, methanesulfonic, ethane disulfonic, oxalic, andisethionic, and the like.

The pharmaceutically acceptable salts of the present invention can besynthesized from the parent compound which contains a basic or acidicmoiety by conventional chemical methods. Generally, such salts can beprepared by reacting the free acid or base forms of these compounds witha stoichiometric amount of the appropriate base or acid in water or inan organic solvent, or in a mixture of the two; generally, nonaqueousmedia like ether, ethyl acetate, ethanol, isopropanol, or acetonitrileare preferred. Lists of suitable salts are found in Remington'sPharmaceutical Sciences, 17th ed., Mack Publishing Company, Easton, Pa.,1985, p. 1418, the disclosure of which is hereby incorporated byreference.

The term “solvate” means a compound, or a salt thereof, that furtherincludes a stoichiometric or non-stoichiometric amount of solvent boundby non-covalent intermolecular forces. Where the solvent is water, thesolvate is a hydrate.

The term “prodrug” means a derivative of a compound that can hydrolyze,oxidize, or otherwise react under biological conditions (in vitro or invivo) to provide an active compound, particularly a compound of theinvention. Examples of prodrugs include, but are not limited to,derivatives and metabolites of a compound of the invention that includebiohydrolyzable moieties such as biohydrolyzable amides, biohydrolyzableesters, biohydrolyzable carbamates, biohydrolyzable carbonates,biohydrolyzable ureides, and biohydrolyzable phosphate analogues.Specific prodrugs of compounds with carboxyl functional groups are thelower alkyl esters of the carboxylic acid. The carboxylate esters areconveniently formed by esterifying any of the carboxylic acid moietiespresent on the molecule. Prodrugs can typically be prepared usingwell-known methods, such as those described by Burger's MedicinalChemistry and Drug Discovery 6th ed. (Donald J. Abraham ed., 2001,Wiley) and Design and Application of Prodrugs (H. Bundgaard ed., 1985,Harwood Academic Publishers Gmfh).

As used herein, and unless otherwise indicated, the terms“biohydrolyzable amide,” “biohydrolyzable ester,” “biohydrolyzablecarbamate,” “biohydrolyzable carbonate,” “biohydrolyzable ureido,”“biohydrolyzable phosphate” mean an amide, ester, carbamate, carbonate,ureido, or phosphate, respectively, of a compound that either: 1) doesnot interfere with the biological activity of the compound but canconfer upon that compound advantageous properties in vivo, such asuptake, duration of action, or onset of action; or 2) is biologicallyinactive but is converted in vivo to the biologically active compound.Examples of biohydrolyzable esters include, but are not limited to,lower alkyl esters, alkoxyacyloxy esters, alkyl acylamino alkyl esters,and choline esters. Examples of biohydrolyzable amides include, but arenot limited to, lower alkyl amides, .alpha.-amino acid amides,alkoxyacyl amides, and alkylaminoalkylcarbonyl amides. Examples ofbiohydrolyzable carbamates include, but are not limited to, loweralkylamines, substituted ethylenediamines, aminoacids,hydroxyalkylamines, heterocyclic and heteroaromatic amines, andpolyether amines.

As used herein, the term “analog thereof” in the context of thecompounds disclosed herein includes diastereomers, hydrates, solvates,salts, prodrugs, and N-oxides of the compounds.

The “translocation domain,” “ligand-binding region”, and chimericreceptors compositions described herein also include “analogs,” or“conservative variants” and “mimetics” (“peptidomimetics”) withstructures and activity that substantially correspond to the exemplarysequences. Thus, the terms “conservative variant” or “analog” or“mimetic” refer to a polypeptide which has a modified amino acidsequence, such that the change(s) do not substantially alter thepolypeptide's (the conservative variant's) structure and/or activity, asdefined herein. These include conservatively modified variations of anamino acid sequence, i.e., amino acid substitutions, additions ordeletions of those residues that are not critical for protein activity,or substitution of amino acids with residues having similar properties(e.g., acidic, basic, positively or negatively charged, polar ornon-polar, etc.) such that the substitutions of even critical aminoacids does not substantially alter structure and/or activity.

More particularly, “conservatively modified variants” applies to bothamino acid and nucleic acid sequences. With respect to particularnucleic acid sequences, conservatively modified variants refers to thosenucleic acids which encode identical or essentially identical amino acidsequences, or where the nucleic acid does not encode an amino acidsequence, to essentially identical sequences. Because of the degeneracyof the genetic code, a large number of functionally identical nucleicacids encode any given protein.

For instance, the codons GCA, GCC, GCG and GCU all encode the amino acidalanine. Thus, at every position where an alanine is specified by acodon, the codon can be altered to any of the corresponding codonsdescribed without altering the encoded polypeptide.

Such nucleic acid variations are “silent variations,” which are onespecies of conservatively modified variations. Every nucleic acidsequence herein which encodes a polypeptide also describes everypossible silent variation of the nucleic acid. One of skill willrecognize that each codon in a nucleic acid (except AUG, which isordinarily the only codon for methionine, and TGG, which is ordinarilythe only codon for tryptophan) can be modified to yield a functionallyidentical molecule. Accordingly, each silent variation of a nucleic acidwhich encodes a polypeptide is implicit in each described sequence.

Conservative substitution tables providing functionally similar aminoacids are well known in the art. For example, one exemplary guideline toselect conservative substitutions includes (original residue followed byexemplary substitution): ala/gly or ser; arg/lys; asn/gln or his;asp/glu; cys/ser; gin/asn; gly/asp; gly/ala or pro; his/asn or gln;ile/leu or val; leu/ile or val; lys/arg or gln or glu; met/leu or tyr orile; phe/met or leu or tyr; ser/thr; thr/ser; trp/tyr; tyr/trp or phe;val/ile or leu. An alternative exemplary guideline uses the followingsix groups, each containing amino acids that are conservativesubstitutions for one another: 1) Alanine (A), Serine (S), Threonine(T); 2) Aspartic acid (D), Glutamic acid (E); 3) Asparagine (N),Glutamine (Q); 4) Arginine (R), Lysine (I); 5) Isoleucine (I), Leucine(L), Methionine (M), Valine (V); and 6) Phenylalanine (F), Tyrosine (Y),Tryptophan (W); (see also, e.g., Creighton, Proteins, W.H. Freeman andCompany (1984); Schultz and Schimer, Principles of Protein Structure,Springer-Verlag (1979)). One of skill in the art will appreciate thatthe above-identified substitutions are not the only possibleconservative substitutions. For example, for some purposes, one mayregard all charged amino acids as conservative substitutions for eachother whether they are positive or negative. In addition, individualsubstitutions, deletions or additions that alter, add or delete a singleamino acid or a small percentage of amino acids in an encoded sequencecan also be considered “conservatively modified variations.”

The terms “mimetic” and “peptidomimetic” refer to a synthetic chemicalcompound that has substantially the same structural and/or functionalcharacteristics of the polypeptides, e.g., translocation domains,ligand-binding regions, or chimeric receptors of the invention. Themimetic can be either entirely composed of synthetic, non-naturalanalogs of amino acids, or may be a chimeric molecule of partly naturalpeptide amino acids and partly non-natural analogs of amino acids. Themimetic can also incorporate any amount of natural amino acidconservative substitutions as long as such substitutions also do notsubstantially alter the mimetic's structure and/or activity.

As with polypeptides of the invention which are conservative variants,routine experimentation will determine whether a mimetic is within thescope of the invention, i.e., that its structure and/or function is notsubstantially altered. Polypeptide mimetic compositions can contain anycombination of non-natural structural components, which are typicallyfrom three structural groups: a) residue linkage groups other than thenatural amide bond (“peptide bond”) linkages; b) non-natural residues inplace of naturally occurring amino acid residues; or c) residues whichinduce secondary structural mimicry, i.e., to induce or stabilize asecondary structure, e.g., a beta turn, gamma turn, beta sheet, alphahelix conformation, and the like. A polypeptide can be characterized asa mimetic when all or some of its residues are joined by chemical meansother than natural peptide bonds. Individual peptidomimetic residues canbe joined by peptide bonds, other chemical bonds or coupling means, suchas, e.g., glutaraldehyde, N-hydroxysuccinimide esters, bifunctionalmaleimides, N,N′-dicyclohexylcarbodiimide (DCC) orN,N′-diisopropylcarbodiimide (DIC). Linking groups that can be analternative to the traditional amide bond (“peptide bond”) linkagesinclude, e.g., ketomethylene (e.g., —C(.═O)—CH₂ for —C(.═O)—NH—),aminomethylene (CH₂NH), ethylene, olefin (CH.dbd.CH), ether (CH₂O),thioether (CH₂—S), tetrazole (CN₄), thiazole, retroamide, thioamide, orester (see, e.g., Spatola, Chemistry and Biochemistry of Amino Acids,Peptides and Proteins, Vol. 7, 267-357, Marcell Dekker, Peptide BackboneModifications, NY (1983)). A polypeptide can also be characterized as amimetic by containing all or some non-natural residues in place ofnaturally occurring amino acid residues; non-natural residues are welldescribed in the scientific and patent literature.

A “label” or a “detectable moiety” is a composition detectable byspectroscopic, photochemical, biochemical, immunochemical, or chemicalmeans. For example, useful labels include ³²P, fluorescent dyes,electron-dense reagents, enzymes (e.g., as commonly used in an ELISA),biotin, digoxigenin, or haptens and proteins which can be madedetectable, e.g., by incorporating a radiolabel into the peptide or usedto detect antibodies specifically reactive with the peptide.

A “labeled nucleic acid probe or oligonucleotide” is one that is bound,either covalently, through a linker or a chemical bond, ornoncovalently, through ionic, van der Waals, electrostatic, or hydrogenbonds to a label such that the presence of the probe may be detected bydetecting the presence of the label bound to the probe.

As used herein a “nucleic acid probe or oligonucleotide” is defined as anucleic acid capable of binding to a target nucleic acid ofcomplementary sequence through one or more types of chemical bonds,usually through complementary base pairing, usually through hydrogenbond formation. As used herein, a probe may include natural (i.e., A, G,C, or T) or modified bases (7-deazaguanosine, inosine, etc.). Inaddition, the bases in a probe may be joined by a linkage other than aphosphodiester bond, so long as it does not interfere withhybridization. Thus, for example, probes may be peptide nucleic acids inwhich the constituent bases are joined by peptide bonds rather thanphosphodiester linkages. It will be understood by one of skill in theart that probes may bind target sequences lacking completecomplementarity with the probe sequence depending upon the stringency ofthe hybridization conditions. The probes are optionally directly labeledas with isotopes, chromophores, lumiphores, chromogens, or indirectlylabeled such as with biotin to which a streptavidin complex may laterbind. By assaying for the presence or absence of the probe, one candetect the presence or absence of the select sequence or subsequence.

The term “heterologous” when used with reference to portions of anucleic acid indicates that the nucleic acid comprises two or moresubsequences that are not found in the same relationship to each otherin nature. For instance, the nucleic acid is typically recombinantlyproduced, having two or more sequences from unrelated genes arranged tomake a new functional nucleic acid, e.g., a promoter from one source anda coding region from another source. Similarly, a heterologous proteinindicates that the protein comprises two or more subsequences that arenot found in the same relationship to each other in nature (e.g., afusion protein).

A “promoter” is defined as an array of nucleic acid sequences thatdirect transcription of a nucleic acid. As used herein, a promoterincludes necessary nucleic acid sequences near the start site oftranscription, such as, in the case of a polymerase II type promoter, aTATA element. A promoter also optionally includes distal enhancer orrepressor elements, which can be located as much as several thousandbase pairs from the start site of transcription. A “constitutive”promoter is a promoter that is active under most environmental anddevelopmental conditions. An “inducible” promoter is a promoter that isactive under environmental or developmental regulation. The term“operably linked” refers to a functional linkage between a nucleic acidexpression control sequence (such as a promoter, or array oftranscription factor binding sites) and a second nucleic acid sequence,wherein the expression control sequence directs transcription of thenucleic acid corresponding to the second sequence.

As used herein, “recombinant” refers to a polynucleotide synthesized orotherwise manipulated in vitro (e.g., “recombinant polynucleotide”), tomethods of using recombinant polynucleotides to produce gene products incells or other biological systems, or to a polypeptide (“recombinantprotein”) encoded by a recombinant polynucleotide. “Recombinant means”also encompass the ligation of nucleic acids having various codingregions or domains or promoter sequences from different sources into anexpression cassette or vector for expression of, e.g., inducible orconstitutive expression of a fusion protein comprising a translocationdomain of the invention and a nucleic acid sequence amplified using aprimer of the invention.

The phrase “selectively (or specifically) hybridizes to” refers to thebinding, duplexing, or hybridizing of a molecule only to a particularnucleotide sequence under stringent hybridization conditions when thatsequence is present in a complex mixture (e.g., total cellular orlibrary DNA or RNA).

The phrase “stringent hybridization conditions” refers to conditionsunder which a probe will hybridize to its target subsequence, typicallyin a complex mixture of nucleic acid, but to no other sequences.Stringent conditions are sequence dependent and will be different indifferent circumstances. Longer sequences hybridize specifically athigher temperatures. An extensive guide to the hybridization of nucleicacids is found in Tijssen, Techniques in Biochemistry and MolecularBiology—Hybridization with Nucleic Probes, “Overview of principles ofhybridization and the strategy of nucleic acid assays” (1993).Generally, stringent conditions are selected to be about 5-10° C. lowerthan the thermal melting point (Tm) for the specific sequence at adefined ionic strength pH. The Tm is the temperature (under definedionic strength, pH, and nucleic concentration) at which 50% of theprobes complementary to the target hybridize to the target sequence atequilibrium (as the target sequences are present in excess, at Tm, 50%of the probes are occupied at equilibrium). Stringent conditions will bethose in which the salt concentration is less than about 1.0 M sodiumion, typically about 0.01 to 1.0 M sodium ion concentration (or othersalts) at pH 7.0 to 8.3 and the temperature is at least about 30° C. forshort probes (e.g., 10 to 50 nucleotides) and at least about 60° C. forlong probes (e.g., greater than 50 nucleotides). Stringent conditionsmay also be achieved with the addition of destabilizing agents such asformamide. For selective or specific hybridization, a positive signal isat least two times background, optionally 10 times backgroundhybridization. Exemplary stringent hybridization conditions can be asfollowing: 50% formamide, 5×SSC, and 1% SDS, incubating at 42° C., or,5×SSC, 1% SDS, incubating at 65° C., with wash in 0.2×SSC, and 0.1% SDSat 65° C. Such hybridizations and wash steps can be carried out for,e.g., 1, 2, 5, 10, 15, 30, 60; or more minutes.

Nucleic acids that do not hybridize to each other under stringentconditions are still substantially related if the polypeptides whichthey encode are substantially related. This occurs, for example, when acopy of a nucleic acid is created using the maximum codon degeneracypermitted by the genetic code. In such cases, the nucleic acidstypically hybridize under moderately stringent hybridization conditions.Exemplary “moderately stringent hybridization conditions” include ahybridization in a buffer of 40% formamide, 1 M NaCl, 1% SDS at 37° C.,and a wash in 1×SSC at 45° C. Such hybridizations and wash steps can becarried out for, e.g., 1, 2, 5, 10, 15, 30, 60, or more minutes. Apositive hybridization is at least twice background. Those of ordinaryskill will readily recognize that alternative hybridization and washconditions can be utilized to provide conditions of similar stringency.

“Antibody” refers to a polypeptide comprising a framework region from animmunoglobulin gene or fragments thereof that specifically binds andrecognizes an antigen. The recognized immunoglobulin genes include thekappa, lambda, alpha, gamma, delta, epsilon, and mu constant regiongenes, as well as the myriad immunoglobulin variable region genes. Lightchains are classified as either kappa or lambda. Heavy chains areclassified as gamma, mu, alpha, delta, or epsilon, which in turn definethe immunoglobulin classes, IgG, IgM, IgA, IgD and IgE, respectively.

An exemplary immunoglobulin (antibody) structural unit comprises atetramer. Each tetramer is composed of two identical pairs ofpolypeptide chains, each pair having one “light” (about 25 kDa) and one“heavy” chain (about 50-70 kDa). The N-terminus of each chain defines avariable region of about 100 to 110 or more amino acids primarilyresponsible for antigen recognition. The terms variable light chain(V_(L)) and variable heavy chain (V_(H)) refer to these light and heavychains respectively.

A “chimeric antibody” is an antibody molecule in which (a) the constantregion, or a portion thereof, is altered, replaced or exchanged so thatthe antigen binding site (variable region) is linked to a constantregion of a different or altered class, effector function and/orspecies, or an entirely different molecule which confers new propertiesto the chimeric antibody, e.g., an enzyme, toxin, hormone, growthfactor, drug, etc.; or (b) the variable region, or a portion thereof, isaltered, replaced or exchanged with a variable region having a differentor altered antigen specificity.

An “anti-T2R” antibody is an antibody or antibody fragment thatspecifically binds a polypeptide encoded by a T2R gene, cDNA, or asubsequence thereof.

The term “immunoassay” is an assay that uses an antibody to specificallybind an antigen. The immunoassay is characterized by the use of specificbinding properties of a particular antibody to isolate, target, and/orquantify the antigen.

The phrase “specifically (or selectively) binds” to an antibody or,“specifically (or selectively) immunoreactive with,” when referring to aprotein or peptide, refers to a binding reaction that is determinativeof the presence of the protein in a heterogeneous population of proteinsand other biologics. Thus, under designated immunoassay conditions, thespecified antibodies bind to a particular protein at least two times thebackground and do not substantially bind in a significant amount toother proteins present in the sample. Specific binding to an antibodyunder such conditions may require an antibody that is selected for itsspecificity for a particular protein.

For example, polyclonal antibodies raised to a T2R family member fromspecific species such as rat, mouse, or human can be selected to obtainonly those polyclonal antibodies that are specifically immunoreactivewith the T2R polypeptide or an immunogenic portion thereof and not withother proteins, except for orthologs or polymorphic variants and allelesof the T2R polypeptide. This selection may be achieved by subtractingout antibodies that cross-react with T2R molecules from other species orother T2R molecules. Antibodies can also be selected that recognize onlyT2R GPCR family members but not GPCRs from other families. A variety ofimmunoassay formats may be used to select antibodies specificallyimmunoreactive with a particular protein. For example, solid-phase ELISAimmunoassays are routinely used to select antibodies specificallyimmunoreactive with a protein (see, e.g., Harlow & Lane, Antibodies, ALaboratory Manual, (1988), for a description of immunoassay formats andconditions that can be used to determine specific immunoreactivity).Typically a specific or selective reaction will be at least twicebackground signal or noise and more typically more than 10 to 100 timesbackground.

The phrase “selectively associates with” refers to the ability of anucleic acid to “selectively hybridize” with another as defined above,or the ability of an antibody to “selectively (or specifically) bind toa protein, as defined above.

The term “expression vector” refers to any recombinant expression systemfor the purpose of expressing a nucleic acid sequence of the inventionin vitro or in vivo, constitutively or inducibly, in any cell, includingprokaryotic, yeast, fungal, plant, insect or mammalian cell. The termincludes linear or circular expression systems. The term includesexpression systems that remain episomal or integrate into the host cellgenome. The expression systems can have the ability to self-replicate ornot, i.e., drive only transient expression in a cell. The term includesrecombinant expression “cassettes which contain only the minimumelements needed for transcription of the recombinant nucleic acid.

By “host cell” is meant a cell that contains an expression vector andsupports the replication or expression of the expression vector. Hostcells may be prokaryotic cells such as E. coli, or eukaryotic cells suchas yeast, insect, amphibian, or mammalian cells such as CHO, HeLa,HEK-293, and the like, e.g., cultured cells, explants, and cells invivo.

Based on the foregoing, the present invention provides assays foridentifying compounds that modulate, preferably block, the specificactivation of the previously identified human bitter taste receptor bybitter compounds, e.g., bitter compounds present in coffee and extractsderived therefrom and structurally related and other bitter compounds.Particularly, the invention provides cell-based assays for identifyingcompounds that modulate (e.g., block) the activation of hT2R8 andhT2R14. These compounds will modulate bitter taste associated with thesetaste receptors in human subjects. This will be confirmed in tastetests.

Also, the invention identifies and provides an antagonist with broadranging antagonist properties that can be used in foods, beverages,medicaments and other materials for human or animal ingestion containingknown and unknown bitter compounds wherein bitter taste is desirablyminimized or eliminated.

That the above taste receptors specifically respond to bittercompound(s) present in coffee and to specific bitter compounds thatinteract with one, multiple, or unknown bitter taste receptors wasdetermined essentially using the HEK293 expression system and calciumimaging methods reported in other publications as well as patentapplications filed by the present Assignee, e.g., U.S. Ser. Nos.10/191,058 and 09/825,882, both incorporated by reference in theirentireties herein. More particularly, the present inventors transfectedHEK293 cells with a particular hT2R tagged with a rhodopsin 35 aminoacid tag (SEQ ID NO:1) together with a chimeric G protein (G16gust44)which comprises the G_(α16) G protein sequence modified by thereplacement of carboxy-44 amino acid residues with those of gustducin,and recorded responses of these cells to specific bitter ligands bycalcium imaging methods.

Specifically, the inventors used a mammalian cell-based assay to monitorhT2R activities. For calcium imaging assays, cells were seeded into48-well tissue culture plates. 24 hours later the cells were transientlytransfected with an expression plasmid (pEAK10) containing an hT2Rnucleic acid sequence, and a plasmid (pEAK10) containing a chimeric Gprotein (G16gust44). Another 24 hours later the cells were incubatedwith a fluorescent dye specific for calcium (Fluo-4; Molecular Probes).The loaded cells are exposed to different bitter molecules, and theactivation of an hT2R leads to activation of G16gust44, which in turnleads to calcium mobilization inside within the cells. This increase incalcium concentration changes the fluorescence properties of the calciumdye inside the cells. These changes are monitored using fluorescencemicroscopy.

The inventors also used the automated fluorimetric aiming system FLIPRusing a slightly different protocol. A HEK293 cell line stablyexpressing G16gust44 was transfected with a hT2R expression plasmid, 24hours later, the cells are loaded and analyzed on FLIPR.

After a ligand is identified for a particular hT2R, a HEK293 cell linestably expressing both the hT2R and G16gust44 are generated,facilitating future screening assays to identify other ligands thatactivate the particular hT2R or which modulate (block or enhance) theactivation of this hT2R by another bitter ligand such as a bittercompound contained in coffee. This avoids the need for transienttransfection.

As shown in the Figures, such experiments revealed that hT2R8 and hT2R14respond to bitter compounds present in coffee and identified compoundsthat inhibit or block the bitter taste of coffee. Also, the experimentsin FIG. 5 and Example 3 infra reveal the broad antagonistic propertiesof Compound C in particular.

These results indicate that cells which identified hT2R taste receptorsmay be used in assays to identify ligands that modulate bitter tasteassociated with at least one of said particular hT2Rs, as well as assaysto detect compounds responsible for bitter taste.

Preferably, these assays will utilize a test cell that expresses a DNAencoding an hT2R having one of the amino acid sequences identifiedinfra. However, it is anticipated that fragments, orthologs, variants orchimeras of these receptor polypeptides which retain the functionalproperties of these bitter taste receptors, i.e., respond to some bittercompounds, will also be useful in these assays. Examples of suchvariants include splice variants, single nucleotide polymorphisms,allelic variants, and mutations produced by recombinant or chemicalmeans, or naturally occurring. Means for isolation and expression ofT2Rs, which are used in the assays of the present invention and assayswhich are contemplated for use in the present invention to identifycompounds that inhibit activation of these receptors, are set forthbelow.

Isolation and Expression of T2Rs

Isolation and expression of the T2Rs, or fragments or variants thereof,of the invention can be effected by well-established cloning proceduresusing probes or primers constructed based on the T2R nucleic acidssequences disclosed in the application. Related T2R sequences may alsobe identified from human or other species genomic databases using thesequences disclosed herein and known computer-based search technologies,e.g., BLAST sequence searching. In a particular embodiment, thepseudogenes disclosed herein can be used to identify functional allelesor related genes.

Expression vectors can then be used to infect or transfect host cellsfor the functional expression of these sequences. These genes andvectors can be made and expressed in vitro or in vivo. One of skill willrecognize that desired phenotypes for altering and controlling nucleicacid expression can be obtained by modulating the expression or activityof the genes and nucleic acids (e.g., promoters, enhancers and the like)within the vectors of the invention. Any of the known methods describedfor increasing or decreasing expression or activity can be used. Theinvention can be practiced in conjunction with any method or protocolknown in the art, which are well described in the scientific and patentliterature.

Alternatively, these nucleic acids can be synthesized in vitro bywell-known chemical synthesis techniques, as described in, e.g.,Carruthers, Cold Spring Harbor Symp. Quant. Biol. 47:411-18 (1982);Adams, Am. Chem. Soc., 105:661 (1983); Belousov, Nucleic Acids Res.25:3440-3444 (1997); Frenkel, Free Radic. Biol. Med. 19:373-380 (1995);Blommers, Biochemistry 33:7886-7896 (1994); Narang, Meth. Enzymol. 68:90(1979); Brown, Meth. Enzymol. 68:109 (1979); Beaucage, Tetra. Lett.22:1859 (1981); U.S. Pat. No. 4,458,066. Double-stranded DNA fragmentsmay then be obtained either by synthesizing the complementary strand andannealing the strands together under appropriate conditions, or byadding the complementary strand using DNA polymerase with an appropriateprimer sequence.

Techniques for the manipulation of nucleic acids, such as, for example,for generating mutations in sequences, subcloning, labeling probes,sequencing, hybridization and the like are well described in thescientific and patent literature. See, e.g., Sambrook, ed., MolecularCloning: A Laboratory Manual (2nd ed.), Vols. 1-3, Cold Spring HarborLaboratory (1989); Ausubel, ed., Current Protocols in Molecular Biology,John Wiley & Sons, Inc., New York (1997); Tijssen, ed., LaboratoryTechniques in Biochemistry and Molecular Biology: Hybridization WithNucleic Acid Probes, Part I, Theory and Nucleic Acid Preparation,Elsevier, N.Y. (1993).

Nucleic acids, vectors, capsids, polypeptides, and the like can beanalyzed and quantified by any of a number of general means well knownto those of skill in the art. These include, e.g., analyticalbiochemical methods such as NMR, spectrophotometry, radiography,electrophoresis, capillary electrophoresis, high performance liquidchromatography (HPLC), thin layer chromatography (TLC), andhyperdiffusion chromatography, various immunological methods, e.g.,fluid or gel precipitin reactions, immunodiffusion,immunoelectrophoresis, radioimmunoassays (RIAs), enzyme-linkedimmunosorbent assays (ELISAs), immuno-fluorescent assays, Southernanalysis, Northern analysis, dot-blot analysis, gel electrophoresis(e.g., SDS-PAGE), RT-PCR, quantitative PCR, other nucleic acid or targetor signal amplification methods, radiolabeling, scintillation counting,and affinity chromatography.

Oligonucleotide primers may be used to amplify nucleic acids encoding aT2R ligand-binding region. The nucleic acids described herein can alsobe cloned or measured quantitatively using amplification techniques.Amplification methods are also well known in the art, and include, e.g.,polymerase chain reaction (PCR) (Innis ed., PCR Protocols, a Guide toMethods and Applications, Academic Press, N.Y. (1990); Innis ed., PCRStrategies, Academic Press, Inc., N.Y. (1995)); ligase chain reaction(LCR) (Wu, Genomics, 4:560 (1989); Landegren, Science, 241:1077 (1988);Barringer, Gene, 89:117 (1990)); transcription amplification (Kwoh,PNAS, 86:1173 (1989)); self-sustained sequence replication (Guatelli,PNAS, 87:1874 (1990)); Q Beta replicase amplification (Smith, J. Clin.Microbiol., 35:1477-91 (1997)); automated Q-beta replicase amplificationassay (Burg, Mol. Cell. Probes, 10:257-71 (1996)); and other RNApolymerase mediated techniques (e.g., NASBA, Cangene, Mississauga,Ontario). See also, Berger, Methods Enzymol., 152:307-16 (1987);Sambrook; Ausubel; U.S. Pat. Nos. 4,683,195 and 4,683,202; Sooknanan,Biotechnology, 13:563-64 (1995).

Once amplified, the nucleic acids, either individually or as libraries,may be cloned according to methods known in the art, if desired, intoany of a variety of vectors using routine molecular biological methods;methods for cloning in vitro amplified nucleic acids are described,e.g., U.S. Pat. No. 5,426,039. To facilitate cloning of amplifiedsequences, restriction enzyme sites can be “built into” the PCR primerpair. For example, Pst I and Bsp E1 sites were designed into theexemplary primer pairs of the invention. These particular restrictionsites have a sequence that, when ligated, are “in-frame” with respect tothe 7-membrane receptor “donor” coding sequence into which they arespliced (the ligand-binding region coding sequence is internal to the7-membrane polypeptide, thus, if it is desired that the construct betranslated downstream of a restriction enzyme splice site, out of frameresults should be avoided; this may not be necessary if the insertedligand-binding region comprises substantially most of the transmembraneVII region). The primers can be designed to retain the original sequenceof the “donor” 7-membrane receptor. Alternatively, the primers canencode amino acid residues that are conservative substitutions (e.g.,hydrophobic for hydrophobic residue, see above discussion) orfunctionally benign substitutions (e.g., do not prevent plasma membraneinsertion, cause cleavage by peptidase, cause abnormal folding ofreceptor, and the like).

The primer pairs may be designed to selectively amplify ligand-bindingregions of T2R proteins. These binding regions may vary for differentligands; thus, what may be a minimal binding region for one ligand, maybe too limiting for a second potential ligand. Thus, binding regions ofdifferent sizes comprising different domain structures may be amplified;for example, transmembrane (TM) domains II through VII, III through VII,III through VI or II through VI, or variations thereof (e.g., only asubsequence of a particular domain, mixing the order of the domains, andthe like), of a 7-transmembrane T2R.

As domain structures and sequence of many 7-membrane T2R proteins areknown, the skilled artisan can readily select domain-flanking andinternal domain sequences as model sequences to design degenerateamplification primer pairs. For example, a nucleic acid sequenceencoding domain regions II through VII can be generated by PCRamplification using a primer pair. To amplify a nucleic acid comprisingtransmembrane domain I (TM I) sequence, a degenerate primer can bedesigned from a nucleic acid that encodes the amino acid sequence of theT2R family consensus sequence 1 described above. Such a degenerateprimer can be used to generate a binding region incorporating TM Ithrough TM III, TM I through TM IV, TM I through TM V, TM I through TMVI or TM I through TM VII). Other degenerate primers can be designedbased on the other T2R family consensus sequences provided herein. Sucha degenerate primer can be used to generate a binding regionincorporating TM III through TM IV, TM III through TM V, TM III throughTM VI or TM III through TM VII.

Paradigms to design degenerate primer pairs are well known in the art.For example, a COnsensus-DEgenerate Hybrid Oligonucleotide Primer(CODEHOP) strategy computer program is accessible ashttp://blocks.fhcrc.org/codehop.html, and is directly linked from theBlockMaker multiple sequence alignment site for hybrid primer predictionbeginning with a set of related protein sequences, as known tastereceptor ligand-binding regions (see, e.g., Rose, Nucleic Acids Res.,26:1628-35 (1998); Singh, Biotechniques, 24:318-19 (1998)).

Means to synthesize oligonucleotide primer pairs are well known in theart. “Natural” base pairs or synthetic base pairs can be used. Forexample, use of artificial nucleobases offers a versatile approach tomanipulate primer sequence and generate a more complex mixture ofamplification products. Various families of artificial nucleobases arecapable of assuming multiple hydrogen bonding orientations throughinternal bond rotations to provide a means for degenerate molecularrecognition. Incorporation of these analogs into a single position of aPCR primer allows for generation of a complex library of amplificationproducts. See, e.g., Hoops, Nucleic Acids Res., 25:4866-71 (1997).Nonpolar molecules can also be used to mimic the shape of natural DNAbases. A non-hydrogen-bonding shape mimic for adenine can replicateefficiently and selectively against a nonpolar shape mimic for thymine(see, e.g., Morales, Nat. Struct. Biol., 5:950-54 (1998)). For example,two degenerate bases can be the pyrimidine base 6H,8H-3,4-dihydropyrimido[4,5-c][1,2]oxazin-7-one or the purine baseN6-methoxy-2,6-diaminopurine (see, e.g., Hill, PNAS, 95:4258-63 (1998)).Exemplary degenerate primers of the invention incorporate the nucleobaseanalog5′-Dimethoxytrityl-N-benzoyl-2′-deoxy-Cytidine,3′-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite(the term “P” in the sequences, see above). This pyrimidine analoghydrogen bonds with purines, including A and G residues.

Polymorphic variants, alleles, and interspecies homologs that aresubstantially identical to a taste receptor disclosed herein can beisolated using the nucleic acid probes described above. Alternatively,expression libraries can be used to clone T2R polypeptides andpolymorphic variants, alleles, and interspecies homologs thereof, bydetecting expressed homologs immunologically with antisera or purifiedantibodies made against a T2R polypeptide, which also recognize andselectively bind to the T2R homolog.

Nucleic acids that encode ligand-binding regions of taste receptors maybe generated by amplification (e.g., PCR) of appropriate nucleic acidsequences using appropriate (perfect or degenerate) primer pairs. Theamplified nucleic acid can be genomic DNA from any cell or tissue ormRNA or cDNA derived from taste receptor-expressing cells.

In one embodiment, hybrid protein-coding sequences comprising nucleicacids encoding T2Rs fused to a translocation sequences may beconstructed. Also provided are hybrid T2Rs comprising the translocationmotifs and taste eliciting compound-binding regions of other families ofchemosensory receptors, particularly taste receptors. These nucleic acidsequences can be operably linked to transcriptional or translationalcontrol elements, e.g., transcription and translation initiationsequences, promoters and enhancers, transcription and translationterminators, polyadenylation sequences, and other sequences useful fortranscribing DNA into RNA. In construction of recombinant expressioncassettes, vectors, and transgenics, a promoter fragment can be employedto direct expression of the desired nucleic acid in all desired cells ortissues.

In another embodiment, fusion proteins may include C-terminal orN-terminal translocation sequences. Further, fusion proteins cancomprise additional elements, e.g., for protein detection, purification,or other applications. Detection and purification facilitating domainsinclude, e.g., metal chelating peptides such as polyhistidine tracts,histidine-tryptophan modules, or other domains that allow purificationon immobilized metals; maltose binding protein; protein A domains thatallow purification on immobilized immunoglobulin; or the domain utilizedin the FLAGS extension/affinity purification system (Immunex Corp,Seattle Wash.).

The inclusion of a cleavable linker sequences such as Factor Xa (see,e.g., Ottavi, Biochimie, 80:289-93 (1998)), subtilisin proteaserecognition motif (see, e.g., Polyak, Protein Eng., 10:615-19(1997));enterokinase (Invitrogen, San Diego, Calif.), and the like, between thetranslocation domain (for efficient plasma membrane expression) and therest of the newly translated polypeptide may be useful to facilitatepurification. For example, one construct can include a polypeptideencoding a nucleic acid sequence linked to six histidine residuesfollowed by a thioredoxin, an enterokinase cleavage site (see, e.g.,Williams, Biochemistry, 34:1787-97 (1995)), and an C-terminaltranslocation domain. The histidine residues facilitate detection andpurification while the enterokinase cleavage site provides a means forpurifying the desired protein(s) from the remainder of the fusionprotein. Technology pertaining to vectors encoding fusion proteins andapplication of fusion proteins are well described in the scientific andpatent literature (see, e.g., Kroll, DNA Cell. Biol., 12:441-53 (1993)).

Expression vectors, either as individual expression vectors or aslibraries of expression vectors, comprising the ligand-binding regionencoding sequences may be introduced into a genome or into the cytoplasmor a nucleus of a cell and expressed by a variety of conventionaltechniques, well described in the scientific and patent literature. See,e.g., Roberts, Nature, 328:731 (1987); Berger supra; Schneider, ProteinExper. Purif., 6435:10 (1995); Sambrook; Tijssen; Ausubel. Productinformation from manufacturers of biological reagents and experimentalequipment also provide information regarding known biological methods.The vectors can be isolated from natural sources, obtained from suchsources as ATCC or GenBank libraries, or prepared by synthetic orrecombinant methods.

The nucleic acids can be expressed in expression cassettes, vectors orviruses which are stably or transiently expressed in cells (e.g.,episomal expression systems). Selection markers can be incorporated intoexpression cassettes and vectors to confer a selectable phenotype ontransformed cells and sequences. For example, selection markers can codefor episomal maintenance and replication such that integration into thehost genome is not required. For example, the marker may encodeantibiotic resistance (e.g., chloramphenicol, kanamycin, G418,bleomycin, hygromycin) or herbicide resistance (e.g., chlorosulfurone orBasta) to permit selection of those cells transformed with the desiredDNA sequences (see, e.g., Blondelet-Rouault, Gene, 190:315-17 (1997);Aubrecht, J. Pharmacol. Exp. Ther., 281:992-97 (1997)). Becauseselectable marker genes conferring resistance to substrates likeneomycin or hygromycin can only be utilized in tissue culture,chemoresistance genes are also used as selectable markers in vitro andin vivo.

A chimeric nucleic acid sequence may encode a T2R ligand-binding regionwithin any 7-transmembrane polypeptide. Because 7-transmembrane receptorpolypeptides have similar primary sequences and secondary and tertiarystructures, structural domains (e.g., extracellular domain, TM domains,cytoplasmic domain, etc.) can be readily identified by sequenceanalysis. For example, homology modeling, Fourier analysis and helicalperiodicity detection can identify and characterize the seven domainswith a 7-transmembrane receptor sequence. Fast Fourier Transform (FFT)algorithms can be used to assess the dominant periods that characterizeprofiles of the hydrophobicity and variability of analyzed sequences.Periodicity detection enhancement and alpha helical periodicity indexcan be done as by, e.g., Donnelly, Protein Sci., 2:55-70 (1993). Otheralignment and modeling algorithms are well known in the art (see, e.g.,Peitsch, Receptors Channels, 4:161-64 (1996); Kyte & Doolittle, J. Md.Biol., 157:105-32 (1982); and Cronet, Protein Eng., 6:59-64 (1993).

The present invention also includes not only the nucleic acid moleculesand polypeptides having the specified nucleic and amino acid sequences,but also fragments thereof, particularly fragments of, e.g., 40, 60, 80,100, 150, 200, or 250 nucleotides, or more, as well as polypeptidefragments of, e.g., 10, 20, 30, 50, 70, 100, or 150 amino acids, ormore. Optionally, the nucleic acid fragments can encode an antigenicpolypeptide that is capable of binding to an antibody raised against aT2R family member. Further, a protein fragment of the invention canoptionally be an antigenic fragment that is capable of binding to anantibody raised against a T2R family member.

Also contemplated are chimeric proteins, comprising at least 10, 20, 30,50, 70, 100, or 150 amino acids, or more, of one of at least one of theT2R polypeptides described herein, coupled to additional amino acidsrepresenting all or part of another GPCR, preferably a member of the 7transmembrane superfamily. These chimeras can be made from the instantreceptors and another GPCR, or they can be made by combining two or moreof the present receptors. In one embodiment, one portion of the chimeracorresponds to, or is derived from the transmembrane domain of a T2Rpolypeptide of the invention. In another embodiment, one portion of thechimera corresponds to, or is derived from the one or more of thetransmembrane regions of a T2R polypeptide described herein, and theremaining portion or portions can come from another GPCR. Chimericreceptors are well known in the art, and the techniques for creatingthem and the selection and boundaries of domains or fragments of GProtein-Coupled Receptors for incorporation therein are also well known.Thus, this knowledge of those skilled in the art can readily be used tocreate such chimeric receptors. The use of such chimeric receptors canprovide, for example, a taste selectivity characteristic of one of thereceptors specifically disclosed herein, coupled with the signaltransduction characteristics of another receptor, such as a well knownreceptor used in prior art assay systems.

For example, a region such as a ligand-binding region, an extracellulardomain, a transmembrane domain, a transmembrane domain, a cytoplasmicdomain, an N-terminal domain, a C-terminal domain, or any combinationthereof, can be covalently linked to a heterologous protein. Forinstance, a T2R transmembrane region can be linked to a heterologousGPCR transmembrane domain, or a heterologous GPCR extracellular domaincan be linked to a T2R transmembrane region. Other heterologous proteinsof choice can include, e.g., green fluorescent protein,.beta.-galactosidase polypeptides, glutamate receptor, and the rhodopsinpolypeptides, e.g., N-terminal fragments of rhodopsin e.g., bovinerhodopsin.

It is also within the scope of the invention to use different host cellsfor expressing the T2Rs, fragments, or variants of the invention. Toobtain high levels of expression of a cloned gene or nucleic acid, suchas cDNAs encoding the T2Rs, fragments, or variants of the invention, oneof skill typically subclones the nucleic acid sequence of interest intoan expression vector that contains a strong promoter to directtranscription, a transcription/translation terminator, and if for anucleic acid encoding a protein, a ribosome binding site fortranslational initiation. Suitable bacterial promoters are well known inthe art and described, e.g., in Sambrook et al. Preferably, eukaryoticexpression systems are used to express the subject hT2R receptor.

Any of the well-known procedures for introducing foreign nucleotidesequences into host cells may be used. These include the use of calciumphosphate transfection, polybrene, protoplast fusion, electroporation,liposomes, microinjection, plasma vectors, viral vectors and any of theother well known methods for introducing cloned genomic DNA, cDNA,synthetic DNA or other foreign genetic material into a host cell (see,e.g., Sambrook et al.) It is only necessary that the particular geneticengineering procedure used be capable of successfully introducing atlest one nucleic acid molecule into the host cell capable of expressingthe T2R, fragment, or variant of interest.

After the expression vector is introduced into the cells, thetransfected cells are cultured under conditions favoring expression ofthe receptor, fragment, or variant of interest, which is then recoveredfrom the culture using standard techniques. Examples of such techniquesare well known in the art. See, e.g., WO 00/06593, which is incorporatedby reference in a manner consistent with this disclosure.

Assays for Detection of Compounds that Modulate the Activity of a hT2RAccording to the Invention

Methods and compositions for determining whether a test compoundspecifically binds to a T2R polypeptide of the invention, both in vitroand in vivo are described below. Many aspects of cell physiology can bemonitored to assess the effect of ligand-binding to a naturallyoccurring or chimeric T2Rs. These assays may be performed on intactcells expressing a T2R polypeptide, on permeabilized cells, or onmembrane fractions produced by standard methods.

Taste receptors bind taste eliciting compounds and initiate thetransduction of chemical stimuli into electrical signals. An activatedor inhibited G protein will in turn alter the properties of targetenzymes, channels, and other effector proteins. Some examples are theactivation of cGMP phosphodiesterase by transducin in the visual system,adenylate cyclase by the stimulatory G protein, phospholipase C by Gqand other cognate G proteins, and modulation of diverse channels by Giand other G proteins. Downstream consequences can also be examined suchas generation of diacyl glycerol and IP3 by phospholipase C, and inturn, for calcium mobilization by IP3.

The subject hT2R proteins or polypeptides of the assay will typically beselected from a polypeptide having a sequence contained in the sequencelisting preceding the claims herein or fragments or conservativelymodified variants thereof.

Alternatively, the T2R proteins or polypeptides of the assay can bederived from a eukaryotic host cell, and can include an amino acidsequence having a certain percentage amino acid sequence identity tothese hT2R polypeptides or conservatively modified variants thereof.Generally, the amino acid sequence identity will be at least 30%preferably 30-40%, more specifically 50-60, 70%, 75%, 80%, 85%, 90%,95%, 96%, 97%, 98%, or 99%. Optionally, the T2R proteins or polypeptidesof the assays can comprise a region of a T2R polypeptide, such as anextracellular domain, transmembrane region, cytoplasmic domain,ligand-binding domain, and the like. Optionally, as exemplified hereinthe T2R polypeptide, or a portion thereof, can be covalently linked to aheterologous protein to create a chimeric protein used in the assaysdescribed herein.

Modulators of T2R activity may be tested using T2R proteins orpolypeptides as described above, either recombinant or naturallyoccurring. The T2R proteins or polypeptides can be isolated, expressedin a cell, expressed in a membrane derived from a cell, expressed intissue or in an animal, either recombinant or naturally occurring. Forexample, tongue slices, dissociated cells from a tongue, transformedcells, or membranes can be used. Modulation can be tested using one ofthe in vitro or in vivo assays described herein.

Detection of Modulators

Compositions and methods for determining whether a test compoundspecifically binds to a T2R receptor of the invention, both in vitro andin vivo, are described below. Many aspects of cell physiology can bemonitored to assess the effect of ligand binding to a T2R polypeptide ofthe invention. These assays may be performed on intact cells expressinga chemosensory receptor, on permeabilized cells, or on membranefractions produced by standard methods or in vitro using de novosynthesized proteins.

In vivo, taste receptors bind to taste modulatory compounds and initiatethe transduction of chemical stimuli into electrical signals. Anactivated or inhibited G protein will in turn alter the properties oftarget enzymes, channels, and other effector proteins. Some examples arethe activation of cGMP phosphodiesterase by transducin in the visualsystem, adenylate cyclase by the stimulatory G protein, phospholipase Cby Gq and other cognate G proteins, and modulation of diverse channelsby Gi and other G proteins. Downstream consequences can also be examinedsuch as generation of diacyl glycerol and IP3 by phospholipase C, and inturn, for calcium mobilization by IP3.

Alternatively, the T2R proteins or polypeptides of the assay can bederived from a eukaryotic host cell and can include an amino acidsubsequence having amino acid sequence identity to the T2R polypeptidesdisclosed herein, or fragments or conservatively modified variantsthereof. Generally, the amino acid sequence identity will be at least 35to 50%, or optionally 75%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%.Optionally, the T2R proteins or polypeptides of the assays can comprisea domain of a T2R protein, such as an extracellular domain,transmembrane region, transmembrane domain, cytoplasmic domain,ligand-binding domain, and the like. Further, as described above, theT2R protein or a domain thereof can be covalently linked to aheterologous protein to create a chimeric protein used in the assaysdescribed herein.

Modulators of T2R receptor activity are tested using T2R proteins orpolypeptides as described above, either recombinant or naturallyoccurring. The T2R proteins or polypeptides can be isolated, expressedin a cell, expressed in a membrane derived from a cell, expressed intissue or in an animal, either recombinant or naturally occurring. Forexample, tongue slices, dissociated cells from a tongue, transformedcells, or membranes can be used. Modulation can be tested using one ofthe in vitro or in vivo assays described herein.

In Vitro Binding Assays

Taste transduction can also be examined in vitro with soluble or solidstate reactions, using the T2R polypeptides of the invention. In aparticular embodiment, T2R ligand-binding domains can be used in vitroin soluble or solid state reactions to assay for ligand binding.

It is possible that the ligand-binding domain may be formed by theN-terminal domain together with additional portions of the extracellulardomain, such as the extracellular loops of the transmembrane domain.

In vitro binding assays have been used with other GPCRs, such as themetabotropic glutamate receptors (see, e.g., Han and Hampson, J. Biol.Chem. 274:10008-10013 (1999)). These assays might involve displacing aradioactively or fluorescently labeled ligand, measuring changes inintrinsic fluorescence or changes in proteolytic susceptibility, etc.

Ligand binding to a T2R polypeptide according to the invention can betested in solution, in a bilayer membrane, optionally attached to asolid phase, in a lipid monolayer, or in vesicles. Binding of amodulator can be tested using, e.g., changes in spectroscopiccharacteristics (e.g., fluorescence, absorbance, refractive index)hydrodynamic (e.g., shape), chromatographic, or solubility properties.

In a preferred embodiment of the invention, a [35S]GTPγS binding assayis used. As described above, upon activation of a GPCR, the Gα subunitof the G protein complex is stimulated to exchange bound GDP for GTP.Ligand-mediated stimulation of G protein exchange activity can bemeasured in a biochemical assay measuring the binding of addedradioactively labeled ^([35S])GTPγS to the G protein in the presence ofa putative ligand. Typically, membranes containing the chemosensoryreceptor of interest are mixed with a G protein. Potential inhibitorsand/or activators and ^([35S])GTPγS are added to the assay, and bindingof ^([35S])GTPγS to the G protein is measured. Binding can be measuredby liquid scintillation counting or by any other means known in the art,including scintillation proximity assays (SPA). In other assays formats,fluorescently labeled GTPγS can be utilized.

Fluorescence Polarization Assays

In another embodiment, Fluorescence Polarization (“FP”) based assays maybe used to detect and monitor ligand binding. Fluorescence polarizationis a versatile laboratory technique for measuring equilibrium binding,nucleic acid hybridization, and enzymatic activity. Fluorescencepolarization assays are homogeneous in that they do not require aseparation step such as centrifugation, filtration, chromatography,precipitation, or electrophoresis. These assays are done in real time,directly in solution and do not require an immobilized phase.Polarization values can be measured repeatedly and after the addition ofreagents since measuring the polarization is rapid and does not destroythe sample. Generally, this technique can be used to measurepolarization values of fluorophores from low picomolar to micromolarlevels. This section describes how fluorescence polarization can be usedin a simple and quantitative way to measure the binding of ligands tothe T2R polypeptides of the invention.

When a fluorescently labeled molecule is excited with plane polarizedlight, it emits light that has a degree of polarization that isinversely proportional to its molecular rotation. Large fluorescentlylabeled molecules remain relatively stationary during the excited state(4 nanoseconds in the case of fluorescein) and the polarization of thelight remains relatively constant between excitation and emission. Smallfluorescently labeled molecules rotate rapidly during the excited stateand the polarization changes significantly between excitation andemission. Therefore, small molecules have low polarization values andlarge molecules have high polarization values. For example, asingle-stranded fluorescein-labeled oligonucleotide has a relatively lowpolarization value but when it is hybridized to a complementary strand,it has a higher polarization value. When using FP to detect and monitortaste eliciting compound-binding which may activate or inhibit thechemosensory receptors of the invention, fluorescence-labeled tasteeliciting compounds or auto-fluorescent taste eliciting compounds may beused.

Fluorescence polarization (P) is defined as:

$P = \underset{\_}{\frac{\left\lbrack {{Int}_{par} - {Int}_{perp}} \right\rbrack}{\left\lbrack {{Int}_{par} + {Int}_{perp}} \right\rbrack}}$

Where Int_(par) is the intensity of the emission light parallel to theexcitation light plane and Int_(perp) is the intensity of the emissionlight perpendicular to the excitation light plane. P, being a ratio oflight intensities, is a dimensionless number. For example, the Beacon™and Beacon 2000™. System may be used in connection with these assays.Such systems typically express polarization in millipolarization units(1 Polarization Unit=1000 mP Units).

The relationship between molecular rotation and size is described by thePerrin equation and the reader is referred to Jolley, M. E. (1991) inJournal of Analytical Toxicology, pp. 236-240 incorporated by reference,which gives a thorough explanation of this equation. Summarily, thePerrin equation states that polarization is directly proportional to therotational relaxation time, the time that it takes a molecule to rotatethrough an angle of approximately 68.5°. Rotational relaxation time isrelated to viscosity (eta.), absolute temperature (T), molecular volume(V), and the gas constant (R) by the following equation: 2(RotationalRelaxation Time)=3 V RT

The rotational relaxation time is small (˜nanosecond) for smallmolecules (e.g. fluorescein) and large (˜100 nanoseconds) for largemolecules (e.g. immunoglobulins). If viscosity and temperature are heldconstant, rotational relaxation time, and therefore polarization, isdirectly related to the molecular volume. Changes in molecular volumemay be due to interactions with other molecules, dissociation,polymerization, degradation, hybridization, or conformational changes ofthe fluorescently labeled molecule. For example, fluorescencepolarization has been used to measure enzymatic cleavage of largefluorescein labeled polymers by proteases, DNases, and RNases. It alsohas been used to measure equilibrium binding for protein/proteininteractions, antibody/antigen binding, and protein/DNA binding.

Solid State and Soluble High Throughput Assays

In yet another embodiment, the invention provides soluble assays using aT2R polypeptide; or a cell or tissue expressing a T2R polypeptide. Inanother embodiment, the invention provides solid phase based in vitroassays in a high throughput format, where the T2R polypeptide, or cellor tissue expressing the T2R polypeptide is attached to a solid phasesubstrate or a taste stimulating compound and contacted with a T2Rreceptor, and binding detected using an appropriate tag or antibodyraised against the T2R receptor.

In the high throughput assays of the invention, it is possible to screenup to several thousand different modulators or ligands in a single day.In particular, each well of a microtiter plate can be used to run aseparate assay against a selected potential modulator, or, ifconcentration or incubation time effects are to be observed, every 5-10wells can test a single modulator. Thus, a single standard microtiterplate can assay about 100 (e.g., 96) modulators. If 1536 well plates areused, then a single plate can easily assay from about 1000 to about 1500different compounds. It is also possible to assay multiple compounds ineach plate well. It is possible to assay several different plates perday; assay screens for up to about 6,000-20,000 different compounds ispossible using the integrated systems of the invention. More recently,microfluidic approaches to reagent manipulation have been developed.

The molecule of interest can be bound to the solid state component,directly or indirectly, via covalent or non-covalent linkage, e.g., viaa tag. The tag can be any of a variety of components. In general, amolecule which binds the tag (a tag binder) is fixed to a solid support,and the tagged molecule of interest (e.g., the taste transductionmolecule of interest) is attached to the solid support by interaction ofthe tag and the tag binder.

A number of tags and tag binders can be used, based upon known molecularinteractions well described in the literature. For example, where a taghas a natural binder, for example, biotin, protein A, or protein G, itcan be used in conjunction with appropriate tag binders (avidin,streptavidin, neutravidin, the Fc region of an immunoglobulin, etc.).Antibodies to molecules with natural binders such as biotin are alsowidely available and appropriate tag binders (see, SIGMA Immunochemicals1998 catalogue SIGMA, St. Louis Mo.).

Similarly, any haptenic or antigenic compound can be used in combinationwith an appropriate antibody to form a tag/tag binder pair. Thousands ofspecific antibodies are commercially available and many additionalantibodies are described in the literature. For example, in one commonconfiguration, the tag is a first antibody and the tag binder is asecond antibody which recognizes the first antibody. In addition toantibody-antigen interactions, receptor-ligand interactions are alsoappropriate as tag and tag-binder pairs. For example, agonists andantagonists of cell membrane receptors (e.g., cell receptor-ligandinteractions such as transferrin, c-kit, viral receptor ligands,cytokine receptors, chemokine receptors, interleukin receptors,immunoglobulin receptors and antibodies, the cadherin family, theintegrin family, the selectin family, and the like; see, e.g., Pigott &Power, The Adhesion Molecule Facts Book I (1993)). Similarly, toxins andvenoms, viral epitopes, hormones (e.g., opiates, steroids, etc.),intracellular receptors (e.g., which mediate the effects of varioussmall ligands, including steroids, thyroid hormone, retinoids andvitamin D; peptides), drugs, lectins, sugars, nucleic acids (both linearand cyclic polymer configurations), oligosaccharides, proteins,phospholipids and antibodies can all interact with various cellreceptors.

Synthetic polymers, such as polyurethanes, polyesters, polycarbonates,polyureas, polyamides, polyethyleneimines, polyarylene sulfides,polysiloxanes, polyimides, and polyacetates can also form an appropriatetag or tag binder. Many other tag/tag binder pairs are also useful inassay systems described herein, as would be apparent to one of skillupon review of this disclosure.

Common linkers such as peptides, polyethers, and the like can also serveas tags, and include polypeptide sequences, such as poly gly sequencesof between about 5 and 200 amino acids. Such flexible linkers are knownto persons of skill in the art. For example, poly(ethylene glycol)linkers are available from Shearwater Polymers, Inc. Huntsville, Ala.These linkers optionally have amide linkages, sulfhydryl linkages, orheterofunctional linkages.

Tag binders are fixed to solid substrates using any of a variety ofmethods currently available. Solid substrates are commonly derivatizedor functionalized by exposing all or a portion of the substrate to achemical reagent which fixes a chemical group to the surface which isreactive with a portion of the tag binder. For example, groups which aresuitable for attachment to a longer chain portion would include amines,hydroxyl, thiol, and carboxyl groups. Aminoalkylsilanes andhydroxyalkylsilanes can be used to functionalize a variety of surfaces,such as glass surfaces. The construction of such solid phase biopolymerarrays is well described in the literature. See, e.g., Merrifield, J.Am. Chem. Soc., 85:2149-2154 (1963) (describing solid phase synthesisof, e.g., peptides); Geysen et al., J. Immun. Meth., 102:259-274 (1987)(describing synthesis of solid phase components on pins); Frank &Doring, Tetrahedron, 44:60316040 (1988) (describing synthesis of variouspeptide sequences on cellulose disks); Fodor et al., Science,251:767-777 (1991); Sheldon et al., Clinical Chemistry, 39(4):718-719(1993); and Kozal et al., Nature Medicine, 2(7):753759 (1996) (alldescribing arrays of biopolymers fixed to solid substrates).Non-chemical approaches for fixing tag binders to substrates includeother common methods, such as heat, cross-linking by UV radiation, andthe like.

Cell-Based Assays

In one preferred embodiment, a T2R protein is expressed in a eukaryoticcell either in unmodified forms or as chimeric, variant or truncatedreceptors with or preferably without a heterologous, chaperone sequencethat facilitates its maturation and targeting through the secretorypathway. Such T2R polypeptides can be expressed in any eukaryotic cell,such as HEK-293 cells. Preferably, the cells comprise a functional Gprotein, e.g., Gα15, or a chimeric Gα16, gustducin or transducin or achimeric G protein such as G16gust44 that is capable of coupling thechimeric receptor to an intracellular signaling pathway or to asignaling protein such as phospholipase C. Activation of T2R receptorsin such cells can be detected using any standard method, such as bydetecting changes in intracellular calcium by detecting FURA-2 dependentfluorescence in the cell. Such an assay is the basis of the experimentalfindings presented in this application.

Activated GPCR receptors often are substrates for kinases thatphosphorylate the C-terminal tail of the receptor (and possibly othersites as well). Thus, activators will promote the transfer of 32P fromradiolabeled ATP to the receptor, which can be assayed with ascintillation counter. The phosphorylation of the C-terminal tail willpromote the binding of arrestin-like proteins and will interfere withthe binding of G proteins. For a general review of GPCR signaltransduction and methods of assaying signal transduction, see, e.g.,Methods in Enzymology, vols. 237 and 238 (1994) and volume 96 (1983);Bourne et al., Nature, 10:349:117-27 (1991); Bourne et al., Nature,348:125-32 (1990); Pitcher et al., Annu. Rev. Biochem., 67:653-92(1998).

T2R modulation may be assayed by comparing the response of T2Rpolypeptides treated with a putative T2R modulator to the response of anuntreated control sample or a sample containing a known “positive”control. Such putative T2R modulators can include molecules that eitherinhibit or activate T2R polypeptide activity. In one embodiment, controlsamples treated with a compound that activates the T2R are assigned arelative T2R activity value of 100. Inhibition of a T2R polypeptide isachieved when the T2R activity value relative to the control sample isabout 90%, optionally 50%, optionally 25-0%. Activation of a T2Rpolypeptide is achieved when the T2R activity value relative to thecontrol is 110%, optionally 150%, 200-500%, or 1000-2000%.

Changes in ion flux may be assessed by determining changes in ionicpolarization (i.e., electrical potential) of the cell or membraneexpressing a T2R polypeptide. One means to determine changes in cellularpolarization is by measuring changes in current (thereby measuringchanges in polarization) with voltage-clamp and patch-clamp techniques(see, e.g., the “cell-attached” mode, the “inside-out” mode, and the“whole cell” mode, e.g., Ackerman et al., New Engl. J Med.,336:1575-1595 (1997)). Whole cell currents are conveniently determinedusing the standard. Other known assays include: radiolabeled ion fluxassays and fluorescence assays using voltage-sensitive dyes (see, e.g.,Vestergarrd-Bogind et al., J. Membrane Biol., 88:67-75 (1988); Gonzales& Tsien, Chem. Biol., 4:269-277 (1997); Daniel et al., J. Pharmacol.Meth., 25:185-193 (1991); Holevinsky et al., J. Membrane Biology,137:59-70 (1994)).

The effects of the test compounds upon the function of the polypeptidescan be measured by examining any of the parameters described above. Anysuitable physiological change that affects GPCR activity can be used toassess the influence of a test compound on the polypeptides of thisinvention. When the functional consequences are determined using intactcells or animals, one can also measure a variety of effects such astransmitter release, hormone release, transcriptional changes to bothknown and uncharacterized genetic markers (e.g., northern blots),changes in cell metabolism such as cell growth or pH changes, andchanges in intracellular second messengers such as Ca2+, IP3, cGMP, orcAMP.

Preferred assays for GPCRs include cells that are loaded with ion orvoltage sensitive dyes to report receptor activity. Assays fordetermining activity of such receptors can also use known agonists andantagonists for other G protein-coupled receptors as controls to assessactivity of tested compounds. In assays for identifying modulatorycompounds (e.g., agonists, antagonists), changes in the level of ions inthe cytoplasm or membrane voltage will be monitored using an ionsensitive or membrane voltage fluorescent indicator, respectively. Amongthe ion-sensitive indicators and voltage probes that may be employed arethose disclosed in the Molecular Probes 1997 Catalog. For Gprotein-coupled receptors, promiscuous G proteins such as Gα15 and Gα16can be used in the assay of choice (Wilkie et al., Proc. Nat'l Acad.Sci., 88:10049-10053 (1991)). Alternatively, other G proteins such asgustducin, transducin and chimeric G proteins such as Gα16gust44 orGalpha16t25 may be used.

Receptor activation initiates subsequent intracellular events, e.g.,increases in second messengers. Activation of some G protein-coupledreceptors stimulates the formation of inositol triphosphate (IP3)through phospholipase C-mediated hydrolysis of phosphatidylinositol(Berridge & Irvine, Nature, 312:315-21 (1984)). IP3 in turn stimulatesthe release of intracellular calcium ion stores. Thus, a change incytoplasmic calcium ion levels, or a change in second messenger levelssuch as IP3 can be used to assess G protein-coupled receptor function.Cells expressing such G protein-coupled receptors may exhibit increasedcytoplasmic calcium levels as a result of contribution from both calciumrelease from intracellular stores and extracellular calcium entry viaplasma membrane ion channels.

In a preferred embodiment, T2R polypeptide activity is measured byexpressing T2R gene in a heterologous cell with a promiscuous G proteinthat links the receptor to a phospholipase C signal transduction pathway(see Offermanns & Simon, J. Biol. Chem., 270:15175-15180 (1995)).Preferably, the cell line is HEK-293 (which does not normally expressT2R genes) and the promiscuous G protein is Gα15 (Offermanns & Simon,supra) or a chimeric G protein such as Gα16gust44. Modulation of tastetransduction is assayed by measuring changes in intracellular Ca2+levels, which change in response to modulation of the T2R signaltransduction pathway via administration of a molecule that associateswith the T2R polypeptide. Changes in Ca2+ levels are optionally measuredusing fluorescent Ca2+ indicator dyes and fluorimetric imaging.

In another embodiment, phosphatidyl inositol (PI) hydrolysis can beanalyzed according to U.S. Pat. No. 5,436,128, herein incorporated byreference. Briefly, the assay involves labeling of cells with3H-myoinositol for 48 or more hrs. The labeled cells are treated with atest compound for one hour. The treated cells are lysed and extracted inchloroform-methanol-water after which the inositol phosphates wereseparated by ion exchange chromatography and quantified by scintillationcounting. Fold stimulation is determined by calculating the ratio of cpmin the presence of agonist, to cpm in the presence of buffer control.Likewise, fold inhibition is determined by calculating the ratio of cpmin the presence of antagonist, to cpm in the presence of buffer control(which may or may not contain an agonist).

Other receptor assays can involve determining the level of intracellularcyclic nucleotides, e.g., cAMP or cGMP. In cases where activation of thereceptor results in a decrease in cyclic nucleotide levels, it may bepreferable to expose the cells to agents that increase intracellularcyclic nucleotide levels, e.g., forskolin, prior to adding areceptor-activating compound to the cells in the assay. In oneembodiment, the changes in intracellular cAMP or cGMP can be measuredusing immunoassays. The method described in Offermanns & Simon, J. Bio.Chem., 270:15175-15180 (1995), may be used to determine the level ofcAMP. Also, the method described in Felley-Bosco et al., Am. J. Resp.Cell and Mol. Biol., 11:159-164 (1994), may be used to determine thelevel of cGMP. Further, an assay kit for measuring cAMP and/or cGMP isdescribed in U.S. Pat. No. 4,115,538, herein incorporated by reference.

In another embodiment, transcription levels can be measured to assessthe effects of a test compound on signal transduction. A host cellcontaining T2R polypeptide of interest is contacted with a test compoundfor a sufficient time to effect any interactions, and then the level ofgene expression is measured. The amount of time to effect suchinteractions may be empirically determined, such as by running a timecourse and measuring the level of transcription as a function of time.The amount of transcription may be measured by using any method known tothose of skill in the art to be suitable. For example, mRNA expressionof the protein of interest may be detected using northern blots or theirpolypeptide products may be identified using immunoassays.Alternatively, transcription based assays using a reporter gene may beused as described in U.S. Pat. No. 5,436,128, herein incorporated byreference. The reporter genes can be, e.g., chloramphenicolacetyltransferase, luciferase, beta-galactosidase, beta-lactamase andalkaline phosphatase. Furthermore, the protein of interest can be usedas an indirect reporter via attachment to a second reporter such asgreen fluorescent protein (see, e.g., Mistili & Spector, NatureBiotechnology, 15:961-964 (1997)).

The amount of transcription is then compared to the amount oftranscription in either the same cell in the absence of the testcompound, or it may be compared with the amount of transcription in asubstantially identical cell that lacks the T2R polypeptide(s) ofinterest. A substantially identical cell may be derived from the samecells from which the recombinant cell was prepared but which had notbeen modified by introduction of heterologous DNA. Any difference in theamount of transcription indicates that the test compound has in somemanner altered the activity of the T2R polypeptide of interest.

Transgenic Non-Human Animals Expressing Chemosensory Receptors

Non-human animals expressing one or more taste receptor sequences of theinvention can also be used for receptor assays. Such expression can beused to determine whether a test compound specifically binds to amammalian taste transmembrane receptor complex in vivo by contacting anon-human animal stably or transiently transfected with nucleic acidsencoding chemosensory receptors or ligand-binding regions thereof with atest compound and determining whether the animal reacts to the testcompound by specifically binding to the receptor polypeptide complex.

Animals transfected or infected with the vectors of the invention areparticularly useful for assays to identify and characterize tastestimuli that can bind to a specific or sets of receptors. Suchvector-infected animals expressing human taste receptor sequences can beused for in vivo screening of taste stimuli and their effect on, e.g.,cell physiology (e.g., on taste neurons), on the CNS, or behavior.

Means to infect/express the nucleic acids and vectors, eitherindividually or as libraries, are well known in the art. A variety ofindividual cell, organ, or whole animal parameters can be measured by avariety of means. The T2R sequences of the invention can be for exampleexpressed in animal taste tissues by delivery with an infecting agent,e.g., adenovirus expression vector.

The endogenous taste receptor genes can remain functional and wild-type(native) activity can still be present. In other situations, where it isdesirable that all taste receptor activity is by the introducedexogenous hybrid receptor, use of a knockout line is preferred. Methodsfor the construction of non-human transgenic animals, particularlytransgenic mice, and the selection and preparation of recombinantconstructs for generating transformed cells are well known in the art.

Construction of a “knockout” cell and animal is based on the premisethat the level of expression of a particular gene in a mammalian cellcan be decreased or completely abrogated by introducing into the genomea new DNA sequence that serves to interrupt some portion of the DNAsequence of the gene to be suppressed. Also, “gene trap insertion” canbe used to disrupt a host gene, and mouse embryonic stem (ES) cells canbe used to produce knockout transgenic animals (see, e.g., Holzschu,Transgenic Res 6:97-106 (1997)). The insertion of the exogenous istypically by homologous recombination between complementary nucleic acidsequences. The exogenous sequence is some portion of the target gene tobe modified, such as exonic, intronic or transcriptional regulatorysequences, or any genomic sequence which is able to affect the level ofthe target gene's expression; or a combination thereof. Gene targetingvia homologous recombination in pluripotential embryonic stem cellsallows one to modify precisely the genomic sequence of interest. Anytechnique can be used to create, screen for, propagate, a knockoutanimal, e.g., see Bijvoet, Hum. Mol. Genet. 7:53-62 (1998); Moreadith,J. Mol. Med. 75:208-216 (1997); Tojo, Cytotechnology 19:161-165 (1995);Mudgett, Methods Mol. Biol. 48:167-184 (1995); Longo, Transgenic Res.6:321-328 (1997); U.S. Pat. Nos. 5,616,491; 5,464,764; 5,631,153;5,487,992; 5,627,059; 5,272,071; WO 91/09955; WO 93/09222; WO 96/29411;WO 95/31560; WO 91/12650.

The nucleic acids of the invention can also be used as reagents toproduce “knockout” human cells and their progeny. Likewise, the nucleicacids of the invention can also be used as reagents to produce“knock-ins” in mice. The human or rat T2R gene sequences can replace theorthologs T2R in the mouse genome. In this way, a mouse expressing ahuman or rat T2R is produced. This mouse can then be used to analyze thefunction of human or rat T2Rs, and to identify ligands for such T2Rs.

Modulators

The compounds tested as modulators of a T2R family member can be anysmall chemical compound, or a biological entity, such as a protein,sugar, nucleic acid or lipid. Alternatively, modulators can begenetically altered versions of a T2R family member. Typically, testcompounds may be small chemical molecules and peptides. Essentially anychemical compound can be used as a potential modulator or ligand in theassays of the invention, although most often compounds can be dissolvedin aqueous or organic (especially DMSO-based) solutions are used. Theassays may be designed to screen large chemical libraries by automatingthe assay steps and providing compounds from any convenient source toassays, which are typically run in parallel (e.g., in microtiter formatson microtiter plates in robotic assays). It will be appreciated thatthere are many suppliers of chemical compounds, including Sigma (St.Louis, Mo.), Aldrich (St. Louis, Mo.), Sigma-Aldrich (St. Louis, Mo.),Fluka Chemika-Biochemica Analytika (Buchs, Switzerland) and the like.

In one embodiment, high throughput screening methods involve providing acombinatorial chemical or peptide library containing a large number ofpotential therapeutic compounds (potential modulator or ligandcompounds). Such “combinatorial chemical libraries” or “ligandlibraries” are then screened in one or more assays, as described herein,to identify those library members (particular chemical species orsubclasses) that display a desired characteristic activity. Thecompounds thus identified can serve as conventional “lead compounds” orcan themselves be used as potential or actual consumer products.

A combinatorial chemical library is a collection of diverse chemicalcompounds generated by either chemical synthesis or biologicalsynthesis, by combining a number of chemical “building blocks” such asreagents. For example, a linear combinatorial chemical library such as apolypeptide library is formed by combining a set of chemical buildingblocks (amino acids) in every possible way for a given compound length(i.e., the number of amino acids in a polypeptide compound). Millions ofchemical compounds can be synthesized through such combinatorial mixingof chemical building blocks.

Preparation and screening of combinatorial chemical libraries is wellknown to those of skill in the art. Such combinatorial chemicallibraries include, but are not limited to, peptide libraries (see, e.g.,U.S. Pat. No. 5,010,175, Furka, Int. J. Pept. Prot. Res., 37:487-93(1991) and Houghton et al., Nature, 354:84-88 (1991)). Other chemistriesfor generating chemical diversity libraries can also be used. Suchchemistries include, but are not limited to: peptoids (e.g., WO91/19735), encoded peptides (e.g., WO 93/20242), random bio-oligomers(e.g., WO 92/00091), benzodiazepines (e.g., U.S. Pat. No. 5,288,514),diversomers such as hydantoins, benzodiazepines and dipeptides (Hobbs etal., PNAS., 90:6909-13 (1993)), vinylogous polypeptides (Hagihara etal., J. Amer. Chem. Soc., 114:6568 (1992)), nonpeptidal peptidomimeticswith glucose scaffolding (Hirschmann et al., J. Amer. Chem. Soc.,114:9217-18 (1992)), analogous organic syntheses of small compoundlibraries (Chen et al., J. Amer. Chem. Soc., 116:2661 (1994)),oligocarbamates (Cho et al., Science, 261:1303 (1993)), peptidylphosphonates (Campbell et al., J. Org. Chem., 59:658 (1994)), nucleicacid libraries (Ausubel, Berger, and Sambrook, all supra), peptidenucleic acid libraries (U.S. Pat. No. 5,539,083), antibody libraries(Vaughn et al., Nature Biotechnology, 14(3):309-14 (1996) andPCT/US96/10287), carbohydrate libraries (Liang et al., Science,274:1520-22 (1996) and U.S. Pat. No. 5,593,853), small organic moleculelibraries (benzodiazepines, Baum, C&EN, January 18, page 33 (1993);thiazolidinones and metathiazanones, U.S. Pat. No. 5,549,974;pynrolidines, U.S. Pat. Nos. 5,525,735 and 5,519,134; morpholinocompounds, U.S. Pat. No. 5,506,337; benzodiazepines, U.S. Pat. No.5,288,514, and the like).

Devices for the preparation of combinatorial libraries are commerciallyavailable (see, e.g., 357 MPS, 390 MPS (Advanced Chem Tech, LouisvilleKy.), Symphony (Rainin, Woburn, Mass.), 433A (Applied Biosystems, FosterCity, Calif.), 9050 Plus (Millipore, Bedford, Mass.)). In addition,numerous combinatorial libraries are themselves commercially available(see, e.g., ComGenex, Princeton, N.J.; Tripos, Inc., St. Louis, Mo.; 3DPharmaceuticals, Exton, Pa.; Martek Biosciences; Columbia, Md.; etc.).

In one aspect of the invention, the T2R modulators can be used in anyfood product, confectionery, pharmaceutical composition, or ingredientthereof to thereby modulate the taste of the product, composition, oringredient in a desired manner. For instance, T2R modulators thatenhance bitter taste sensation can be added to provide a bitter taste toa product or composition, while T2R modulators which block bitter tastesensations can be added to block the bitter taste of a product orcomposition. Also, the invention provides means of identifying bittercompounds found in foods, beverages, cosmetics and medicinals andproducing taste improved foods, beverages and medicinals lacking orhaving a reduced quantity thereof.

Use of Compounds Identified by the Invention

Compounds identified according to the invention may be added to foods,beverages, cosmetics or medicinal compositions to modulate, preferablyblock bitter taste triggered by activation at least one of one of hT2R8and/or hT2R14 by bitter compounds present in coffee and related foods,beverages and medicaments or structurally related compounds or otherbitter compounds, e.g., compounds found in foods and beverages ormedicinals or cosmetics that elicit a bitter taste perception.

In particular Compound C, and its analogs, based on its broad rangingantagonist properties may be used as an additive in any food, beverage,medicament or material for consumption by humans or animals whereinbitter taste is desirably alleviated. Given Compound C's properties,these materials may contain bitter ligands known to interact withspecific bitter ligands such as hT2R3, 7, 10, 14, 16, 44, 51, 55, 61,63, 64, 65, or 71 and/or with hT2R5, 9, 13, 54, 67 and 75, or acombination thereof or which contain bitter compounds for which theirbitter receptor selectivity is undetermined. Especially preferredapplications are compositions containing compounds that activatemultiple bitter taste receptors.

In addition, the subject compounds including Compound C may be used incompetitive binding and functional assays as well as taste tests toidentify bitter compounds for which Compound C blocks or inhibits bittertaste.

As noted previously, preferably, the taste modulatory properties,preferably bitter taste blocking properties of compounds identified inthe subject T2R cell-based assays will be confirmed in human or animaltaste tests, preferably human taste tests.

Kits

T2R genes and their homologs are useful tools for identifying tastereceptor cells, for forensics and paternity determinations, and forexamining taste transduction. T2R family member-specific reagents thatspecifically hybridize to T2R nucleic acids, such as T2R probes andprimers, and T2R specific reagents that specifically bind to a T2Rprotein, e.g., T2R antibodies are used to examine taste cell expressionand taste transduction regulation.

Nucleic acid assays for the presence of DNA and RNA for a T2R familymember in a sample include numerous techniques are known to thoseskilled in the art, such as southern analysis, northern analysis, dotblots, RNase protection, S1 analysis, amplification techniques such asPCR, and in situ hybridization. In in situ hybridization, for example,the target nucleic acid is liberated from its cellular surroundings insuch as to be available for hybridization within the cell whilepreserving the cellular morphology for subsequent interpretation andanalysis. The following articles provide an overview of the art of insitu hybridization: Singer et al., Biotechniques, 4:230250 (1986); Haaseet al., Methods in Virology, vol. VII, 189-226 (1984); and Names et al.,eds., Nucleic Acid Hybridization: A Practical Approach (1987). Inaddition, a T2R protein can be detected with the various immunoassaytechniques described above. The test sample is typically compared toboth a positive control (e.g., a sample expressing a recombinant T2Rprotein) and a negative control.

The present invention also provides for kits for screening formodulators of T2R family members. Such kits can be prepared from readilyavailable materials and reagents. For example, such kits can compriseany one or more of the following materials: T2R nucleic acids orproteins, reaction tubes, and instructions for testing T2R activity.Optionally, the kit contains a functional T2R polypeptide. A widevariety of kits and components can be prepared according to the presentinvention, depending upon the intended user of the kit and theparticular needs of the user.

Having now generally described the invention, the same will be morereadily understood by reference to the following examples, which areprovided by way of illustration and are not intended as limiting. It isunderstood that various modifications and changes can be made to theherein disclosed exemplary embodiments without departing from the spiritand scope of the invention.

EXAMPLES Example 1

hT2R8 and hT2R14 are Activated by Bitter Coffee Fraction

A partially purified bitter fraction from coffee was used to screen the25 human T2Rs in transiently transfected HEK cells as described inprevious patent applications. In brief (as discussed in more detail inU.S Patent Publication No. 2003/0170608, incorporated herein byreference), human embryonic kidney cells that stably express largeT-cell antigen and G15 protein (HEK-G15) were transiently transfectedwith an hT2R expression plasmid (e.g., by use of Ca²⁺ phosphate or byuse of lipid-based systems). Additionally, other HEK-G15 cell lines weretransiently transfected with other human T2Rs. Thereafter, afluorescent-based assay was used to detect changes in calciumconcentration in the transiently transfected cells. Interaction of thetest compound(s) with the transfected cells elicits a signaling cascadeleading to the activation of PLC and a subsequent increase inintracellular calcium concentration resulting in an increase offluorescence which was detected using a calcium-sensitive fluorescentdye. These changes were monitored e.g., using fluorescence microscopicand appropriately designed software (such as Imaging Workstation, Axon).

The coffee fraction has high level of fluorescence, which interferedwith the assay. To overcome the interference, a number of blue dyes weretested for ability to block the fluorescence from the coffee fraction.As shown in FIG. 1, the coffee fraction activated hT2R8 and hT2R14 incalcium imaging assay using transiently transfected cells. The blue dyeused in the experiment of FIG. 1 is FD&C 1 at 1.9 mM. Several otherhT2Rs also appeared to be activated by this coffee fraction. Withdifferent blue dyes, different combinations of hT2Rs are activated(Table 1). However, hT2R8 and hT2R14 are consistently picked up asresponsive to the coffee fraction, and the activities of these tworeceptors are coffee fraction dose-dependent (FIG. 2) The blue dye usedin the experiment of FIG. 2 was tryptan blue.

TABLE 1 hT2Rs activated by the coffee fraction with different blue dyeshT2R Receptors Identified Blue Dye Activate Weakly activate FD&C 1 8, 14— Trypan 1, 8, 14 10, 75 Coomassie 14 —

Using this assay, it was found that the addition of the bitter fractionfrom coffee to cells that express hT2R8 and hT2R14 activatedintracellular G proteins. By contrast, using the same assay, the bitterfraction from coffee did not specifically activate HEK-G15 cells thatwere transiently transfected with other hT2Rs. This experiment supportsthe conclusion that taste receptors hT2R8 and hT2R14 specificallyrespond to bitter compound(s) present in coffee.

Example 2

Identification of Antagonists of hT2R8 and hT2R14

To identify antagonists, cell lines stably expressing hT2R8 and hT2R14,respectively, together with the promiscuous chimeric G16g44 protein weregenerated as described in previous patent applications. Ahigh-throughput assay was established using the stable cell lines andFLIPR (Fluorescent Imaging Plate Reader). An agonist of hT2R8 or hT2R14was used to activate the receptors up to 70-80% of their respectivemaximal activity. For hT2R8, the agonist used was andrographolide (200μM); for hT2R14, it was aristolochic acid (3 μM). To identifyantagonists compounds with diverse chemical structures were addedtogether with the agonist. Compounds that cause statisticallysignificant reduction of the receptor activity are pooled together, andreconfirmed with dose-dependent inhibition curves. Compound A andCompound B were identified as hT2R8 antagonists (FIG. 3). Compound C wasidentified as an hT2R14 antagonist (FIG. 4).

Example 2a

Combinations hT2R8 and hT2R14 Antagonists Reduce Bitter Taste of Coffee

Taste tests were performed with combinations of hT2R8 and hT2R14antagonists in the coffee fraction and two types of instant coffee(medium roast and medium-dark roast), using a 2-alternative forcedchoice method with a taste panel of 4-5 panelists. Coffee samples withthe antagonists were given to the taste panelists together with the samesample without antagonists, the panelists were asked to identify thebitterer sample within the pair. As shown in Table 2, the panelistsconsistently identified the coffee fraction samples without antagonistsas being bitterer than the ones with antagonists, indicating that theantagonists reduced the bitter taste of the coffee fraction. Similarly,as shown in Table 3, the antagonists reduced the bitter taste of bothtypes of instant coffee.

As demonstrated by the taste tests of this example, the perception ofbitterness in compositions (e.g., foods, beverages and/or medicaments)which exhibit bitter taste may be reduced or eliminated by incorporationof antagonists of hT2R8 and/or hT2R14 into such compositions.

To determine the contribution of an individual antagonist, taste testswere performed with medium roast instant coffee with Compound C. Asshown in Table 4, the hT2R14 antagonist (Compound C) is sufficient byitself to reduce the bitter taste in the coffee of this example.

TABLE 2 Taste test results with coffee fraction and 2 differentcombinations of antagonists Selected as bitterer AntagonistConcentration Without With Test hT2R8 hT2R14 (μM) antagonistsantagonists P value 1 Cmp A Cmp C 30 32 0 <0.001 2 Cmp B Cmp C 30 15 10.001 3 Cmp A Cmp C 10 16 0 <0.001

TABLE 3 Taste test results with 2 types of instant coffee Concen-Selected as bitterer Instant Antagonists tration Without Plus P CoffeehT2R8 hT2R14 (μM) antagonists antagonists value Medium Cmp A Cmp C 30 160 <0.001 Medium- Cmp A Cmp C 30 13 3 0.021 dark

TABLE 4 Taste test results with medium roast coffee and individualantagonist Selected as bitterer Taste Concentration Without With TestAntagonist (μM) antagonists Antagonist P value 1 Compound C 50 18 2<0.001 2 Compound C 25 19 1 <0.001

Example 3

Compound C is a Broadly Acting Bitter Receptor Antagonist

Example 2 above teaches that compound C is a human T2R antagonistidentified by high throughput screening assays using hT2R14. Additionalexperiments reveal that Compound C is a broadly tuned antagonist for 13human T2Rs and to a lesser extent antagonizes six other human T2Rs.Moreover, this compound in taste tests blocks the bitter taste intensityelicited by a number of diverse bitter substances.

Specifically, in order to evaluate the inhibitory selectivity of theCompound C compound, this compound was tested against 22 human T2Rswhich were deorphaned by Senomyx. These receptors and the bitter ligandsthat activate these human T2Rs are reported in earlier patentapplications which are incorporated by reference herein. These 22 humanT2Rs are hT2R1, 3, 4, 5, 7, 8, 9, 10, 13, 14, 16, 44, 51, 54, 55, 61,63, 64, 65, 67, 71 and 75. The amino acid and nucleic acid sequences ofall of these T2Rs may be found in these earlier patent applications.These human T2Rs were each individually transiently transfected intoHEK293 cells that stably express the promiscuous G protein G16g44 andfunctional assays were effected using these receptors as disclosed inthese same patent applications. In these experiments each receptor wasactivated by one of its ligands selected from bitter moleculespreviously demonstrated to activate the particular T2R. The ligands wereused at EC80 concentration levels. The list of bitter ligands utilizedand the tested ligand concentrations are contained in Table 5 of thisexample.

Furthermore, in order to confirm the in vitro activity of this compoundin the receptor assay, the inventors performed paired comparison tastetest to determine the effect of the compound in vivo. The tastepanelists were asked to taste bitter substances with and withoutCompound C, and to identify which sample tastes more bitter. Multiplepairs were tasted by each panelist to increase the sample size, and theresults were analyzed using appropriate statistical methods. The orderof samples with and without Compound C were randomized and counterbalanced.

In order to establish the broad antagonistic properties of thiscompound, it was tested for its ability to block bitter taste elicitedby a variety of bitter ligands as well bitter taste elicited by bitterligands known to activate multiple bitter taste receptors and bitterligands which have not yet been demonstrated to activate a specifichT2R. Several bitter molecules known to activate bitter receptors weretested for which activation is inhibited by Compound C. Specifically,salicin is a bitter molecule that activates hT2R16, and taste testresults showed that Compound C at 40 μM can reduce its bitter taste.Phenylthiourea is a bitter molecule that activates hT2R51, and CompoundC reduced its bitter taste at 25 μM.

Several bitter molecules that can activate multiple T2Rs were similarlytested with Compound C. The activation of bitter receptors for some ofthese molecules were partially inhibited by Compound C. Omeprazole is abitter molecule that activates hT2R10, 14 and 75. Notwithstanding thatits bitter taste may involve multiple bitter receptors, its bitter tastewas also appreciably reduced by Compound C. Rebaudioside A is a naturalsweetener with strong bitter taste, which activates at least 7 humanT2Rs. Its bitter taste is also reduced by Compound C.

In addition, Compound C inhibited bitter taste for some compoundswherein the receptor(s) with which they interact is unknown, such asdextromethorphan and diphenhydramin. The effect of Compound C on thesecompounds was tested and it was discovered that their bitter taste wasalso reduced.

Relating to the foregoing, FIG. 5 contains experimental results whereinCompound C was tested with different agonist compounds. The inhibitionactivity is represented by the reduction of receptor activity in thepresence of Compound C. FIG. 5 reveals that 13 different hT2Rs weresignificantly (greater than 30%) inhibited by Compound C. These 13 hT2Rsare hT2R3, 7, 10, 14, 16, 44, 51, 55, 61, 63, 64, 65, and 71. Six otherreceptors, including hT2R5, 9, 13, 54, 67 and 75 were also inhibited,although to a lesser extent.

TABLE 5 List of ligands and concentrations used for each tested T2R.hT2Rs Agonist Concentration 1 Picric Acid 0.05 mM 3 Chloroquine pH 6.550 μM 4 Chloroquine pH 6.5 5 mM 5 Picoline 10 mM 7 Chloroquine pH 6.5 10mM 8 Andrographolide 0.5 mM 9 Ofloxacin 1 mM 10 Strychnine 50 μM 13Oxyphenonium 1 mM 14 Aristolochic Acid 2 μM 16 Salicin 1 mM 44DenatoniμM 0.5 μM 51 Prop 2.5 μM 54 Ranitidine 5 mM 55 Cinchonine 150 μM61 Aristolochic Acid 25 nM 63 Caffeine 1 mM 63 Andrographolide 100 μM 64Aristolochic Acid 1 μM 65 Oleuropein 1 mM 67 Andrographolide 5 μM 71Picric Acid 10 μM 75 Strychnine 1 μM

Example 4

hT2R8 Antagonists: Making the Compounds of the Invention

Exemplary Compounds According to the Invention are Synthesized asFollows

Example 4-1N-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)picolinamide

1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-amine hydrochloride(example 4-1a) (400 mg, 2.1 mmol), picolinic acid (256 mg, 2.1 mmol),and HOBt (388 mg, 2.50 mmol) were mixed in DCM (7 mL). The reaction wastreated with triethylamine (670 mL, 4.8 mmol) and stirred for 15 minutesat room temperature under a nitrogen atmosphere. EDC (598 mg, 3.1 mmol)was added and the reaction was stirred for an additional 4 hours. Thereaction was then diluted with dichloromethane (5 mL) and washed withaqueous saturated NaHCO₃ solution (5 mL, 2×) and then with aqueoussaturated NaCl solution (5 mL). The organic layer was collected, dried,and filtered. Solvents were removed under vacuum. The crude product wasre-suspended in EtOH (5 mL) and purified by reversed phase HPLC (5%-95%ACN in H₂O: 25 minute gradient). The pure fractions were combined andconcentrated to affordN-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)picolinamide(372 mg, 60%) as a white solid. ¹H NMR (CDCl₃, 400 MHz): δ 2.21 (s, 3H),2.44 (s, 3H), 5.05 (s, 2H), 7.49-7.47 (m, 1H), 7.59 (s, 1H), 7.93-7.88(dt, J=14, 2 Hz, 1H), 8.07 (s, 1H), 8.24-8.21 (d, J=8 Hz, 1H), 8.61-8.56(m, 1H), 9.83 (bs, 1H). LC/MS; [M+H] calculated for C15H15N5O2; expected297.1; found 298.3. Melting point: 135-137° C.

The compound had an IC₅₀ on hT2R8 bitter receptor of 0.57 μM

Example 4-1a 1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-aminehydrochloride

Tert-butyl1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-ylcarbamate (Example4-1b) (592 mg, 2 mmol) was stirred in a solution of 4N HCl in dioxane(20 mL) at ambient temperature for 2 hours. The solvent was removedunder reduced pressure and the residue was taken up in a 1/1 mixture ofethyl acetate/hexanes (30 mL) and concentrated (twice). The solid wastriturated with hexanes and collected by filtration providing1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-amine hydrochloride(500 mg, 99%) as a white solid. ¹H NMR (DMSO-d₆, 400 MHz):

2.11 (s, 3H), 2.38 (s, 3H), 5.16 (s, 2H), 7.51 (s, 1H), 8.03 (s, 1H),10.27 (bs, 3H).

Example 4-1b tert-butyl1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-ylcarbamate

3,5-dimethyl-4-((4-nitro-1H-pyrazol-1-yl)methyl)isoxazole (Example 4-1c)(14.6 g, 66 mmol), Boc anhydride, and 10% Pd/C (3.8 g) was stirred inMeOH (400 mL) under 1 atmosphere of H₂ for 16 hours at ambienttemperature. The mixture was filtered and the solution was removed underreduced pressure. The residue was purified by silica gel chromatography(20% ethyl acetate in hexanes) to afford tert-butyl1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-ylcarbamate (12.7 g,66%) as a light pink solid. ¹H NMR (CDCl₃, 400 MHz):

1.41 (s, 9H), 2.10 (s, 3H), 2.32 (s, 3H), 4.90 (s, 2H), 6.19 (bs, 1H),7.19 (s, 1H), 7.50 (s, 1H). MS 293 (MH⁺).

Example 4-1c 3,5-dimethyl-4-((4-nitro-1H-pyrazol-1-yl)methyl)isoxazole

To 4-nitro-1H-pyrazole (Example 4-1d) (3.8 g, 34 mmol) in DMF (80 mL)cooled to 0° C., via an ice/water bath, was added t-BuOK (4.2 g, 38mmol). After addition of the base the ice bath was removed and themixture was stirred for 30 minutes followed by the addition of4-(chloromethyl)-3,5-dimethylisoxazole (5 g, 34 mmol). The reaction wasrefluxed for 16 hours then cooled to ambient temperature. H₂O was addedto the reaction mixture and the precipitate formed was collected byfiltration. The precipitate was washed with additional H₂O then driedunder high vacuum to afford3,5-dimethyl-4-((4-nitro-1H-pyrazol-1-yl)methyl)isoxazole (5.8 g, 78%)as a light yellow solid. ¹H NMR (CDCl₃, 400 MHz):

2.23 (s, 3H), 2.46 (s, 3H), 5.08 (s, 2H), 8.02 (s, 1H), 8.08 (s, 1H).

Example 4-1d 4-nitro-1H-pyrazole

Pyrazole (10 g, 147 mmol), was added to concentrated sulfuric acid (100mL), in portions, while maintaining the internal reaction temperaturebelow 50° C. via an ice water bath. Concentrated nitric acid (10 mL) wasthen added, dropwise, maintaining the internal reaction temperaturebelow 50° C. via an ice water bath. The ice water bath was removed andthe reaction was heated to 60° C. and stirred for 4 hours. The reactionwas cooled via an ice water bath and made basic, to ˜pH 8, with 18 Naqueous NaOH solution (150 mL). The product, which precipitated as awhite solid, was collected by filtration, washed with H₂O, and driedunder high vacuum to afford 4-nitro-1H-pyrazole (7 g, 42%) as a whitesolid. ¹³C NMR (100 MHz, CDCl₃): δ 126.4, 137.0.

Example 4-23-chloro-N-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-4-(methylsulfonyl)thiophene-2-carboxamide

To a stirring mixture of((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-amine hydrochloride(Example 4-1a) (500 mg, 2 mmol) in DCM (20 mL), cooled to 0° C. via anice water bath, was added triethylamine (600 mg, 6 mmol). The mixturewas stirred until all solids were in solution (˜10 minutes).3-Chloro-4-(methylsulfonyl)thiophene-2-carbonyl chloride (543 mg, 2.1mmol), in 2 mL CH₃CN, was added via syringe to the free amine at 0° C.The ice bath was removed and the mixture was stirred for 2 hours. Thereaction was diluted with dichloromethane (100 mL) and the organic phasewas washed with H₂O (200 mL). The organic layer was dried over sodiumsulfate, filtered and concentrated under reduced pressure. The solid wastriturated with ethyl acetate/hexanes (1/5) to afford3-chloro-N-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-4-(methylsulfonyl)thiophene-2-carboxamide(375 mg, 45%) as a white solid. ¹H NMR (CDCl₃, 400 MHz):

2.20 (s, 3H), 2.43 (s, 4H), 3.22 (s, 3H), 5.05 (s, 2H), 7.57 (s, 1H),7.94 (s, 1H), 8.41 (s, 1H), 8.59 (bs, 1H). LC/MS; [M+H] 415.5. Meltingpoint: 202-204° C.

The compound had an IC₅₀ on hT2R8 bitter receptor of 2.09 μM

Example 4-3(S)-N-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-2-phenylpropanamide

1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-amine hydrochloride(Example 4-1a) (200 mg, 1 mmol), (S)-2-phenyl propionic acid (156 mg, 1mmol), and PyBop (650 mg, 1.3 mmol) were added to DMF (4 mL) followed bytriethylamine (0.3 mL, 2.1 mmol). The reaction stirred for 4 hours atroom temperature under a nitrogen atmosphere then diluted with ethylacetate (20 mL), washed with aqueous saturated NaHCO₃ solution (2×15 mL)followed by aqueous saturated NaCl solution (15 mL). The organic phasewas dried, filtered and concentrated on the rotovap. The crude productwas re-suspended in methanol (3 mL) and purified by reversed phase HPLC(5%-95% ACN in H₂O: 25 minute gradient). The fractions containing thepure product were concentrated to afford(S)-N-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-2-phenylpropanamide(200 mg, 60%) as a white solid. ¹H NMR (400 MHz, DMSO-d₆) δ 1.36 (d,J=7.2, Hz, 3H), 2.09 (s, 3H), 2.36 (s, 3H), 3.71-3.66 (m, 1H), 5.05 (s,2H), 7.33-7.17 (m, 5H), 7.37 (s, 1H), 7.90 (s, 1H), 10.05 (s, 1H). MS325 (M+H). Melting point 108° C.-110° C.

The compound had an IC₅₀ on hT2R8 bitter receptor of 0.41 μM

Example 4-4N-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-2-(4-hydroxy-3,5-dimethoxyphenyl)acetamide

1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-amine hydrochloride(Example 4-1a) (376 mg, 1.7 mmol),2-(4-hydroxy-3,5-dimethoxyphenyl)acetic acid (350 mg, 1.7 mmol), PyBop(1 g, 2 mmol) and triethylamine (605 mg, 6 mmol) were stirred togetherin DMF (10 mL) at room temperature for 2 hours. The reaction mixture wasdiluted with aqueous 1N HCl (100 mL) and extracted with DCM (3×, 75 mL).The combined organic extracts were dried over sodium sulfate, filteredand concentrated on the rotovap. The residue was purified by silica gelchromatography (30% ethyl acetate in hexanes) to affordN-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-2-(4-hydroxy-3,5-dimethoxyphenyl)acetamide(189 mg, 29%) as a white solid. ¹H NMR (CDCl₃, 400 MHz)

2.10 (s, 3H), 2.36 (s, 3H), 3.40 (s, 2H), 3.70 (s, 6H), 5.07 (s, 2H),6.53 (s, 2H), 7.39 (s, 1H), 7.92 (s, 1H), 8.18 (s, 1H), 10.03 (s, 1H).LC/MS; [M+H] calculated for C19H22N4O5; expected 387.16; found 387.6.Melting point: 187-188° C.

The compound had an IC₅₀ on hT2R8 bitter receptor of 0.46 μM

Example 4-5N-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-2-phenylpropanamide

1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-amine hydrochloride(Example 4-1a) (300 mg, 1.3 mmol), 2-phenylpropanoic acid (225 mg, 1.5mmol), triethylamine (300 mg, 3 mmol), DMAP (61 mg, 0.5 mmol), and EDC(386 mg, 2 mmol) were stirred together in DCM (10 mL) at roomtemperature for 4 hours. The reaction mixture was diluted with aqueous1N HCl (100 mL) and extracted with DCM (3×, 75 mL). The combined organicextracts were dried over sodium sulfate, filtered and concentrated, onthe rotovap. The residue was purified by silica gel chromatography (30%ethyl acetate in hexanes) to affordN-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-2-phenylpropanamide(272 mg, 81%) as a white solid. ¹H NMR (CDCl₃, 400 MHz) δ 1.36 (d, 3H,J=7.2 Hz), 2.10 (s, 3H), 2.37 (s, 3H), 3.70 (m, 1H, J=6.8 Hz), 5.06 (s,2H), 7.20 (t, 1H, J=8.4 Hz), 7.31-7.28 (m, 4H), 7.38 (s, 1H), 7.91 (s,1H), 10.10 (s, 1H). LC/MS; [M+H] calculated for C18H20N4O2; expected325.16; found 325.5. Melting point: 129-130° C.

The compound had an IC₅₀ on hT2R8 bitter receptor of 0.32 μM

Example 4-6N-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-2-phenylacetamide

1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-amine hydrochloridesalt (Example 4-1a) (230 mg, 1 mmol) and triethylamine (300 mg, 3 mmol)were stirred in DCM (10 mL) cooled to 0° C. via an ice/water bath.2-Phenylacetyl chloride (184 mg, 1.3 mmol) was added dropwise to thestirring reaction mixture. When the addition was complete the ice bathwas removed and the reaction was stirred for 1 hour. The mixture wasdiluted with DCM (50 mL), washed with 1N aqueous HCl (100 mL), followedby 1N aqueous NaOH (100 mL) and then H₂O (100 mL). The combined organicextracts were dried over sodium sulfate, filtered and the solvent wasremoved on the rotovap. The resulting residue was purified by silica gelchromotography (50% ethyl acetate in hexanes) to afford 210 mg of solidproduct which was triturated in ethyl acetate/hexanes (1/9) to affordN-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-2-phenylacetamide(188 mg, 68%) as a white solid. ¹H NMR (CDCl₃, 400 MHz):

2.15 (s, 3H), 2.38 (s, 3H), 3.69 (s, 2H), 4.97 (s, 2H), 7.15 b(s, 1H),7.40-7.27 (m, 6H), 7.84 (s, 1H). LC/MS; [M+H] calculated for C17H18N4O2;expected 311.14; found 311.40. Melting point: 106-108° C.

The compound had an IC₅₀ on hT2R8 bitter receptor of 0.53 μM

Example 4-7N-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-3-methoxybenzamide

1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-amine hydrochloride(Example 4-1a) (300 mg, 1.3 mmol), 3-methoxybenzoic acid (172 mg, 1.3mmol), EDC (386 mg, 2 mmol), and triethyl amine (303 mg, 3 mmol) werestirred in DCM (5 mL) at ambient temperature for 6 hours. The reactionwas diluted with DCM (50 mL) and the organic phase was washed withaqueous 0.1 N HCL (150 mL) followed by aqueous 1N NaOH (150 mL). Theorganic layer was dried over sodium sulfate, filtered and concentratedon the rotovap. The crude product was purified by silica gelchromotography (40% ethyl acetate in hexanes) to afford 225 mg of an offwhite solid. The solid was triturated with ethyl acetate/hexanes (1/9)and the white solid was collect by filtration. The pure product wasdissolved in absolute ethanol and concentrated on the rotovap (4×, 25mL) to affordN-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-3-methoxybenzamide(185 mg, 43%) as a white solid. ¹H NMR (CDCl₃, 400 MHz):

2.20 (s, 3H), 2.42 (s, 3H), 3.85 (s, 3H), 5.03 (s, 2H), 7.09-7.06 (m,1H), 7.37-7.35 (m, 2H), 7.41 (m, 1H), 7.51 (s, 1H), 7.93 (bs, 1H), 8.03(s, 1H). LC/MS; [M+H] calculated for C17H18N4O3; expected 327.14; found327.30. Melting point: 127-129° C.

The compound had an IC₅₀ on hT2R8 bitter receptor of 0.39 μM

Example 4-8N-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)benzo[d][1,3]dioxole-5-carboxamide

1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-amine hydrochloride(example 1a) (8 mg, 35 μmol) and benzo[d][1,3]dioxole-5-carboxylic acid(7 mg, 42 μmol) were each dissolved in 200 uL dimethylformamide.Si-Carbodiimide resin (70 mg, 70 μmol) was loaded into a 1.2 mL 96 wellGreiner plate, followed by the addition of amine and acid.Hydroxybenzotriazole (6 mg, 42 μmol) was dissolved in 100 uLdimethylformamide and was added into the reaction well. The reaction wasshaken overnight at room temperature. To remove excess carboxylic acidand hydroxybenzotriazole, PS-Trisamine resin (35 mg, 70 μmol) was addedinto the reaction mixture and was allowed to shake overnight at roomtemperature. 200 uL of Acetonitrile was added into the reaction well andshaken for 1 minute. The top clear solution was transferred into a newplate. The extraction process was repeated two more times. The solutionwas evaporated under vacuum and gave the desired product. Yield 6%. MSM+H calculated 341.1, found 341.2.

The compound had an IC₅₀ on hT2R8 bitter receptor of 0.2 μM

Example 4-9N-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-2,5-dimethoxybenzamide

Prepared as in Example 4-8 from 2,5-dimethoxybenzoic acid and1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-amine hydrochloride(Example 4-1a). Yield 13%. MS M+H calculated 357.5, found 357.3.

The compound had an IC₅₀ on hT2R8 bitter receptor of 0.17 μM

Example 4-103-cyano-N-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)benzamide

Prepared as in Example 4-8 from 3-cyanobenzoic acid and1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-amine hydrochloride(Example 4-1a). Yield 15%. MS M+H calculated 322.6, found 322.3.

The compound had an IC₅₀ on hT2R8 bitter receptor of 0.2 μM

Example 4-11N-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-1-phenylcyclopropanecarboxamide

Prepared as in Example 4-8 from 1-phenylcyclopropanecarboxylic acid and1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-amine hydrochloride(Example 4-1a). Yield 6%. MS M+H calculated 337.6, found 337.5.

The compound had an IC₅₀ on hT2R8 bitter receptor of 0.25 μM

Example 4-12N-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-3-phenylbutanamide

Prepared as in Example 4-8 from 3-phenylbutanoic acid and1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-amine hydrochloride(Example 4-1a). Yield 6%. MS M+H calculated 339.6, found 339.5.

The compound had an IC₅₀ on hT2R8 bitter receptor of 0.28 μM

Example 4-13N-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-1H-pyrrole-2-carboxamide

Prepared as in Example 4-8 from 1H-pyrrole-2-carboxylic acid and1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-amine hydrochloride(Example 4-1a). Yield 18%. MS M+H calculated 286.6, found 286.3.

The compound had an IC₅₀ on hT2R8 bitter receptor of 0.57 μM

Example 4-142-cyclohexyl-N-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)acetamide

Prepared as in Example 4-8 from 2-cyclohexylacetic acid and1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-amine hydrochloride(Example 4-1a). Yield 17%. MS M+H calculated 317.6, found 317.4.

The compound had an IC₅₀ on hT2R8 bitter receptor of 0.73 μM

Example 4-15N-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)cinnamamide

Prepared as in Example 4-8 from cinnamic acid and1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-amine hydrochloride(Example 4-1a). Yield 4%. MS M+H calculated 322.6, found 322.4.

The compound had an IC₅₀ on hT2R8 bitter receptor of 0.7 μM

Example 4-16N-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)adamantane

1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-amine hydrochloride(300 mg, 1.56 mmol), adamantane-1-carboxylic acid (281 mg, 1.56 mmol),PyBop (972 mg, 1.87 mmol), and triethylamine (0.438 mL, 3.12 mmol) weremixed in DMF (5 mL). The reaction stirred at room temperature for 4hours under a nitrogen atmosphere. The reaction was diluted with ethylacetate (4 mL) and washed with saturated NaHCO₃ solution (2×, 3 mL) andthen with saturated NaCl solution (3 mL). The organic layer wasextracted, dried, and filtered. Solvents were removed under vacuum. Thecrude product was re-suspended in methanol (4 mL) and purified by HPLC.The pure product was re-dissolved in ethanol and concentrated undervacuum (3×3 mL) to affordN-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl) adamantane asa white solid in 60% yield. ¹H NMR (400 MHz, CDCl₃):

1.79-1.70{tilde over (.)}(m, 6H), 1.93-1.92 (m, 6H), 2.08 (bs, 3H), 2.18(s, 3H), 2.41 (s, 3H), 4.98 (s, 2H), 7.37 (s, 1H), 7.38 (s, 1H), 7.92(s, 1H). MS 355 (M+H). Melting point 167-169° C.

The compound had an IC₅₀ on hT2R8 bitter receptor of 0.88 μM

Additional compounds were synthesized following similar procedures asdescribed in examples 4-1 to 4-16 and experimentally tested and found tohave a relatively high level of effectiveness as inhibitors of hT2R8bitter receptor. The results of that testing are shown below in Table A.

TABLE A Compound hT2R8 No. Compound IC₅₀ (μM) 4-17

N-(1-((3,5-dimethylisoxazol-4- yl)methyl)-1H-pyrazol-4-yl)-4-methoxybenzamide 0.26 4-18

N-(1-((3,5-dimethylisoxazol-4- yl)methyl)-1H-pyrazol-4-yl)-3,5-dimethoxybenzamide 0.28 4-19

N-(1-((3,5-dimethylisoxazol-4- yl)methyl)-1H-pyrazol-4-yl)-2-methoxybenzamide 0.39 4-20

N-(1-((3,5-dimethylisoxazol-4- yl)methyl)-1H-pyrazol-4-yl)-4-hydroxy-3-methoxybenzamide 0.48 4-21

N-(1-((3,5-dimethylisoxazol-4- yl)methyl)-1H-pyrazol-4-yl)-3,4-dimethoxybenzamide 0.56 4-23

N-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-4-fluorobenzamide 1.56 4-24

N-(1-((3,5-dimethylisoxazol-4- yl)methyl)-1H-pyrazol-4-yl)benzamide 2.624-25

N-(1-((3,5-dimethylisoxazol-4- yl)methyl)-1H-pyrazol-4-yl)-2,3-dimethoxybenzamide 0.61 4-26

N-(1-((3,5-dimethylisoxazol-4- yl)methyl)-1H-pyrazol-4-yl)-3-(methylsulfonyl)benzamide 0.72 4-27

N-(1-((3,5-dimethylisoxazol-4- yl)methyl)-1H-pyrazol-4-yl)-2-hydroxy-5-methoxybenzamide 0.98 4-28

N-(1-((3,5-dimethylisoxazol-4- yl)methyl)-1H-pyrazol-4-yl)-3-methylbenzamide 0.57 4-29

N-(1-((3,5-dimethylisoxazol-4- yl)methyl)-1H-pyrazol-4-yl)-2-methoxybenzamide 0.30 4-30

N-(1-((3,5-dimethylisoxazol-4- yl)methyl)-1H-pyrazol-4-yl)-2-methoxybenzamide 0.39 4-31

2-(2,3-dimethoxyphenyl)-N-(1-((3,5- dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)acetamide 0.80 4-32

2-(3,5-dimethoxyphenyl)-N-(1-((3,5- dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)acetamide 0.96 4-33

N-(1-((3,5-dimethylisoxazol-4- yl)methyl)-1H-pyrazol-4-yl)-2-(4-methoxyphenyl)acetamide 0.99 4-34

N-(1-((3,5-dimethylisoxazol-4- yl)methyl)-1H-pyrazol-4-yl)-2-methoxybenzamide 1.11 4-35

N-(1-((3,5-dimethylisoxazol-4- yl)methyl)-1H-pyrazol-4-yl)-2-(2-methoxyphenyl)acetamide 1.16 4-36

N-(1-((3,5-dimethylisoxazol-4- yl)methyl)-1H-pyrazol-4-yl)-2-methoxybenzamide 1.85 4-37

N-(1-((3,5-dimethylisoxazol-4- yl)methyl)-1H-pyrazol-4-yl)-2-phenylbutanamide 0.50 4-38

N-(1-((3,5-dimethylisoxazol-4- yl)methyl)-1H-pyrazol-4-yl)-2-methoxybenzamide 0.53 4-39

2-(1-((3,5-dimethylisoxazol-4- yl)methyl)-1H-pyrazol-4-ylamino)-2-oxo-1-phenylethyl acetate 0.83 4-40

N-(1-((3,5-dimethylisoxazol-4- yl)methyl)-1H-pyrazol-4-yl)-3-hydroxy-2-phenylpropanamide 0.96 4-41

N-(1-((3,5-dimethylisoxazol-4- yl)methyl)-1H-pyrazol-4-yl)-2-methoxybenzamide 2.10 4-42

(R)-N-(1-((3,5-dimethylisoxazol-4- yl)methyl)-1H-pyrazol-4-yl)-2-phenylpropanamide 3.72 4-43

N-(1-((3,5-dimethylisoxazol-4- yl)methyl)-1H-pyrazol-4-yl)-2-methoxy-2-phenylacetamide 3.43 4-44

N-(1-((3,5-dimethylisoxazol-4- yl)methyl)-1H-pyrazol-4-yl)-2,2-diphenylacetamide 5.19 4-45

N-(1-((3,5-dimethylisoxazol-4- yl)methyl)-1H-pyrazol-4-yl)-4-methyl-1H-pyrrole-2-carboxamide 0.72 4-46

N-(1-((3,5-dimethylisoxazol-4- yl)methyl)-1H-pyrazol-4-yl)nicotinamide1.05 4-47

N-(1-((3,5-dimethylisoxazol-4- yl)methyl)-1H-pyrazol-4-yl)-2-methoxybenzamide 1.35 4-48

N-(1-((3,5-dimethylisoxazol-4- yl)methyl)-1H-pyrazol-4-yl)-6-methylpicolinamide 1.95 4-49

N-(1-((3,5-dimethylisoxazol-4- yl)methyl)-1H-pyrazol-4-yl)-1-methyl-1H-pyrrole-2-carboxamide 3.40 4-50

N-(1-((3,5-dimethylisoxazol-4- yl)methyl)-1H-pyrazol-4-yl)isonicotinamide 4.59 4-51

N-(1-((3,5-dimethylisoxazol-4- yl)methyl)-1H-pyrazol-4-yl)-2-methylfuran-3-carboxamide 7.87 4-52

N-(1-((3,5-dimethylisoxazol-4- yl)methyl)-1H-pyrazol-4-yl)-1,3-dimethyl-1H-pyrazole-5-carboxamide 18.05 4-53

N-(1-((3,5-dimethylisoxazol-4- yl)methyl)-1H-pyrazol-4-yl)-2,5-dimethylfuran-3-carboxamide 3.03 4-54

N-(1-((3,5-dimethylisoxazol-4- yl)methyl)-1H-pyrazol-4-yl)-5-methylfuran-2-carboxamide 3.19 4-55

N-(1-((3,5-dimethylisoxazol-4- yl)methyl)-1H-pyrazol-4-yl)-3-phenylpropanamide 0.49 4-56

N-(1-((3,5-dimethylisoxazol-4- yl)methyl)-1H-pyrazol-4-yl)-4-phenylbutanamide 0.83 4-57

N-(1-((3,5-dimethylisoxazol-4- yl)methyl)-1H-pyrazol-4-yl)-2-methoxybenzamide 4.16 4-58

N-(1-((3,5-dimethylisoxazol-4- yl)methyl)-1H-pyrazol-4-yl)-2-(furan-3-yl)acetamide 8.66 4-59

N-(1-((3,5-dimethylisoxazol-4- yl)methyl)-1H-pyrazol-4-yl)-3-ethoxybenzamide 0.22 4-60

N-(1-((3,5-dimethylisoxazol-4- yl)methyl)-1H-pyrazol-4-yl)-3-methylpentanamide 1.94 4-61

N-(1-((3,5-dimethylisoxazol-4- yl)methyl)-1H-pyrazol-4-yl)cyclohexanecarboxamide 2.20 4-62

N-(1-((3,5-dimethylisoxazol-4- yl)methyl)-1H-pyrazol-4-yl)-2-(adamanth-1-yl)acetamide 3.77 4-63

1-((3,5-dimethylisoxazol-4-yl)methyl)- N-methyl-1H-pyrazol-4-amine 8.764-64

N,N-bis(2,3-dimethoxybenzyl)-1-((3,5- dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-amine 12.60 4-65

N-(1-((3,5-dimethylisoxazol-4- yl)methyl)-1H-pyrazol-4-yl)-N-methyl-2-phenylpropanamide 2.81 4-66

N-(1-((3,5-dimethylisoxazol-4- yl)methyl)-1H-pyrazol-4-yl)-N-methyl-1-phenylcyclopropanecarboxamide 8.23 4-67

(S)-N-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-2-methoxy- 2-phenylacetamide 8.6 4-68

(R)-N-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-2-methoxy- 2-phenylacetamide 5.0 4-69

N-(1-((3,5-dimethylisoxazol-4- yl)methyl)-1H-pyrazol-4-yl)-2-(4-hydroxy-3-methoxyphenyl)acetamide 4.7 4-70

N-(1-((3,5-dimethylisoxazol-4- yl)methyl)-1H-pyrazol-4-yl)-2-(3-hydroxy-4-methoxyphenyl)acetamide 3.1 4-71

N-(1-((3,5-dimethylisoxazol-4- yl)methyl)-1H-pyrazol-4-yl)-2-hydroxy-3-methoxybenzamide 2.9 4-72

N-(1-((3,5-dimethylisoxazol-4- yl)methyl)-1H-pyrazol-4-yl)benzo[b]oxazole-5-carboxamide 2.6 4-73

N-(1-((3,5-dimethylisoxazol-4- yl)methyl)-1H-pyrazol-4-yl)-3,4-dihydroxy-5-methoxybenzamide 2.2 4-74

N-(1-((3,5-dimethylisoxazol-4- yl)methyl)-1H-pyrazol-4-yl)-3-methoxy-4-methylbenzamide 2.2 4-75

N-(1-((3,5-dimethylisoxazol-4- yl)methyl)-1H-pyrazol-4-yl)-2-methylbenzofuran-5-carboxamide 2.1 4-76

N-(1-((3,5-dimethylisoxazol-4- yl)methyl)-1H-pyrazol-4-yl)quinoxaline-5-carboxamide 1.7 4-77

N-(1-((3,5-dimethylisoxazol-4- yl)methyl)-1H-pyrazol-4-yl)benzofuran-5-carboxamide 1.4 4-78

N-(1-((3,5-dimethylisoxazol-4- yl)methyl)-1H-pyrazol-4-yl)-2,2-difluorobenzo[d][1,3]dioxole-5- carboxamide 1.3 4-79

N-(1-((3,5-dimethylisoxazol-4- yl)methyl)-1H-pyrazol-4-yl)-2-methyl-1H-benzo[d]imidazole-5-carboxamide 1.0 4-80

3-chloro-N-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)benzamide 0.9 4-81

N-(1-((3,5-dimethylisoxazol-4- yl)methyl)-1H-pyrazol-4-yl)-2-ethylbenzo[d]oxazole-5-carboxamide 0.8 4-82

N-(1-((3,5-dimethylisoxazol-4- yl)methyl)-1H-pyrazol-4-yl)quinoxaline-6-carboxamide 0.8 4-83

N-(1-((3,5-dimethylisoxazol-4- yl)methyl)-1H-pyrazol-4-yl)-2-methylbenzo[d]oxazole-6-carboxamide 0.7 4-85

N-(1-((3,5-dimethylisoxazol-4- yl)methyl)-1H-pyrazol-4-yl)-1H-benzo[d]imidazole-5-carboxamide 0.6 4-86

2-(1H-benzo[d]imidazol-6-yl)-N-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H- pyrazol-4-yl)acetamide 0.6 4-87

N-(1-((3,5-dimethylisoxazol-4- yl)methyl)-1H-pyrazol-4-yl)-1H-benzo[d][1,2,3]triazole-5-carboxamide 0.6 4-88

N-(1-((3,5-dimethylisoxazol-4- yl)methyl)-1H-pyrazol-4-yl)-2,3-dihydrobenzo[b][1,4]dioxine-6-carboxamide 0.4 4-89

7-bromo-N-(1-((3,5-dimethylisoxazol-4- yl)methyl)-1H-pyrazol-4-yl)-2,3-dihydrobenzo[b][1,4]dioxine-6-carboxamide 0.3 4-90

7-chloro-N-(1-((3,5-dimethylisoxazol-4- yl)methyl)-1H-pyrazol-4-yl)-2,3-dihydrobenzo[b][1,4]dioxine-6-carboxamide 0.1 4-91

8-chloro-N-(1-((3,5-dimethylisoxazol-4- yl)methyl)-1H-pyrazol-4-yl)-2,3-dihydrobenzo[b][1,4]dioxine-6-carboxamide 0.1 4-96

1-((3,5-dimethylisoxazol-4-yl)methyl)- N-methyl-1H-pyrazol-4-amine 8.7644-97

N-(1-((3,5-dimethylisoxazol-4- yl)methyl)-1H-pyrazol-4-yl)-N-methyl-2-phenylpropanamide 2.126 4-98

(S)-N-(1-((3,5-dimethylisoxazol-4- yl)methyl)-1H-pyrazol-4-yl)-N-methyl-2-phenylpropanamide 2.811 4-99

2-cyano-N-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)benzenesulfonamide 1.358 4-100

N-(1-((3,5-dimethylisoxazol-4- yl)methyl)-1H-pyrazol-4-yl)-2-fluorobenzenesulfonamide 8.510 4-101

N-(1-((3,5-dimethylisoxazol-4- yl)methyl)-1H-pyrazol-4-yl)-2,5-dimethoxybenzenesulfonamide 1.631 4-102

N-(1-((3,5-dimethylisoxazol-4- yl)methyl)-1H-pyrazol-4-yl)-2-methylbenzenesulfonamide 2.153 4-103

N-(1-((3,5-dimethylisoxazol-4- yl)methyl)-1H-pyrazol-4-yl)-2-methoxybenzenesulfonamide 3.801 4-104

methyl 3-(N-(1-((3,5-dimethylisoxazol- 4-yl)methyl)-1H-pyrazol-4-yl)sulfamoyl)hiophene-2-carboxylate 1.252 4-105

1-(1-((3,5-dimethylisoxazol-4- yl)methyl)-1H-pyrazol-4-yl)-3-(2-fluorophenyl)thiourea 1.629 4-106

1-(1-((3,5-dimethylisoxazol-4- yl)methyl)-1H-pyrazol-4-yl)-3-(2-methoxyphenyl)thiourea 2.607 4-107

1-(1-((3,5-dimethylisoxazol-4- yl)methyl)-1H-pyrazol-4-yl)-3-(pyridin-3-yl)thiourea 2.999 4-108

1-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-3-o-tolylthiourea 3.013 4-109

1-(3-cyanophenyl)-3-(1-((3,5- dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)thiourea 0.783 4-110

1-benzyl-3-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)thiourea 1.097 4-111

1-(2-cyanophenyl)-3-(1-((3,5- dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)thiourea 2.347 4-112

1-(1-((3,5-dimethylisoxazol-4- yl)methyl)-1H-pyrazol-4-yl)-3-phenylthiourea 2.492 4-113

1-(2,5-dimethoxyphenyl)-3-(1-((3,5- dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)thiourea 5.240 4-114

N-(2-((3,5-dimethylisoxazol-4- yl)methyl)-2H-tetrazol-5-yl)-3-methoxybenzamide 1.866 4-115

N-(1-((3,5-dimethylisoxazol-4- yl)methyl)-3-methyl-1H-pyrazol-4-yl)-3-methoxybenzamide 9.248 4-116

3-((3,5-dimethylisoxazol-4-yl)methyl)-2-oxo-N-(thiophen-2-ylmethyl)-2,3- dihydrothiazole-5-carboxamide 2.279

Examples 4-67 to 4-91

Prepared as in example 4-73 from1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-amine hydrochloride(example 4-1a) and their corresponding functionalized carboxylic acids.Characterization was done by LCMS where the desired masses were found.

Example 4-73N-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-3,4-dihydroxy-5-methoxybenzamide

1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-amine hydrochloride(example 4-1a) (228 mg, 1 mmol), 3,4-dihydroxy-5-methoxybenzoic acid(184 mg, 1 mmol), HOBt (135 mg, 1 mmol), and EDC (191 mg, 1 mmol) weredissolved in 2 mL DMF in a microwave vial followed by the addition oftriethylamine (101 mg, 1 mmol). The reaction was placed in a microwavereactor at 165° C. for 5 minutes. The crude product was purifieddirectly using Varian HPLC (10%-95% ACN in H₂O: 25 minute gradient). Thepure fractions were combined and concentrated to affordN-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-3,4-dihydroxy-5-methoxybenzamide.(280 mg, 70%). LC/MS; [M+H] calculated for C17H18N4O5; expected 359.1;found 359.1.

The compound had an IC₅₀ on hT2R8 bitter receptor of 2.2 μM

Example 4-92N-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-7-methoxybenzo[d][1,3]dioxole-5-carboxamide

N-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-3,4-dihydroxy-5-methoxybenzamide(example 73) (50 mg, 0.14 mmol) and Cesium Carbonate (113 mg, 2.5 mmol)were dissolved in 1 mL acetone followed by the addition ofdibromomethane (239 mg, 1.4 mmol). The reaction was placed in amicrowave reactor at 120° C. for 20 minutes. The clear solution from thereaction was removed and evaporated under vacuum. The crude product wasdissolved in 1 mL ethanol and purified by varian HPLC (10%-95% ACN inH₂O: 25 minute gradient). The pure fractions were combined andconcentrated to affordN-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-2-(7-methoxybenzo[d][1,3]dioxol-5-yl)acetamide.(12 mg, 23%). LC/MS; [M+H] calculated for C18H18N4O5; expected 371.1;found 371.1.

The compound had an IC₅₀ on hT2R8 bitter receptor of 0.7 μM

Example 4-93N-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-8-methoxy-2,3-dihydrobenzo[b][1,4]dioxine-6-carboxamide

Prepared as in Example 4-92 fromN-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-3,4-dihydroxy-5-methoxybenzamide(Example 4-73), cesium carbonate and dibromoethane. Yield 20%. MS M+Hcalculated 385.1, found 385.1.

The compound had an IC₅₀ on hT2R8 bitter receptor of 0.7 μM

Example 4-94N-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-7-methoxy-2-methylbenzo[d][1,3]dioxole-5-carboxamide

Prepared as in Example 4-92 fromN-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-3,4-dihydroxy-5-methoxybenzamide(Example 4-73), cesium carbonate and 1,1-dibromoethane. Yield 25%. MSM+H calculated 385.1, found 385.1.

The compound had an IC₅₀ on hT2R8 bitter receptor of 0.7 μM.

Example 4-95N-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-7-methoxy-2,3-dihydrobenzo[b][1,4]dioxine-6-carboxamide

Prepared as in Example 4-73 from1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-amine hydrochloride(example 4-1a) and7-methoxy-2,3-dihydrobenzo[b][1,4]dioxine-6-carboxylic acid (example4-95a). Yield 50%. MS M+H calculated 385.1, found 385.1. ¹H NMR (400MHz, DMSO): 2.136 (s, 3H), 2.410 (s, 3H), 3.851 (s, 3H), 4.214 (bs, 2H),4.296 (bs, 2H), 5.120 (s, 2H), 6.688 (s, 1H), 7.290 (s, 1H), 7.6006 (s,1H), 8.069 (s, 1H), 9.856 (s, 1H).

The compound had an IC₅₀ on hT2R8 bitter receptor of 0.7 μM

Example 4-95a 7-Methoxy-2,3-dihydrobenzo[b][1,4]dioxine-6-carboxylicacid

In a 2 mL microwave vial, methyl7-bromo-2,3-dihydrobenzo[b][1,4]dioxine-6-carboxylate (273 mg, 1 mmol)and CuBr (14.3 mg, 0.1 mmol) were dissolved in dry DMF and was placed inice bath. Sodium methoxide (540 mg, 10 mmol) was added into the reactionmixture dropwise while stirring at 0° C. The reaction was warmed to roomtemperature and stirred for 45 minutes. The reaction was then placed ina microwave reactor for 5 minutes at 135° C. The reaction mixture wasdissolved in water and washed with ethyl acetate. The water layer wascollected and acidified to pH 4 with 1M HCl. The product was extractedusing ethyl acetate then dried over sodium sulfate. The solvent wasevaporated under vacuum to give the desired intermediate of7-methoxy-2,3-dihydrobenzo[b][1,4]dioxine-6-carboxylic acid which wasused directly without further purification. Yield 57%. MS M+H calculated211.1, found 211.1.

Example 5 hT2R14 Antagonists: Making the Compounds of the Invention

The following Examples are given to illustrate a variety of exemplaryembodiments of the invention and are not intended to be limiting in anymatter.

Example 5-1 4-(N-benzyl-N-(4-methoxybenzyl)sulfamoyl)benzoic acid

Benzyl 4-(N-benzyl-N-(4-methoxybenzyl)sulfamoyl)benzoate (Example 5-1a)(517 mg, 1 mmol) was stirred a 10/1/2 solution of 6N NaOH(aq)/THF/MeOH(27 mL) at ambient temperature for 6 hours. The solution was acidifiedwith 3N HCl (aq) to a pH of ˜3 (ca. 50 mL), and the aqueous phase wasextracted with ethyl acetate (3×, 75 mL). The combined organic extractswere dried over sodium sulfate and concentrated on the rotovap. Theresidue was taken up in MeOH (15 mL) and purified by reverse phased HPLC(5-95% acetonitrile in H₂O gradient: 25 minutes) in three 5 mL aliquots.The pure fractions were combined and concentrated to a white solid. Theproduct was dissolved in 15 mL of absolute ethanol and evaporated on therotovap (4×) to provide pure4-(N-benzyl-N-(4-methoxybenzyl)sulfamoyl)benzoic acid (174 mg, 42%) as awhite solid. M.p 161-163° C. ¹H NMR (CDCl₃, 400 MHz):

3.78 (s, 3H), 4.32 (s, 2H), 4.36 (s, 2H), 6.78 (d, J=8.4 Hz, 2H), 6.99(d, J=8.8 Hz, 2H), 709-7.07 (m, 2H), 7.27-7.24 (m, 3H), 7.93 (d, J=8.8Hz, 2H), 8.24 (d, J=8 Hz, 2H). MS 412 (MH⁺).

The compound had an IC₅₀ on hT2R14 bitter receptor of 0.22 μM

Example 5-1a Benzyl 4-(N-benzyl-N-(4-methoxybenzyl)sulfamoyl)benzoate

4-(N-(4-methoxybenzyl)sulfamoyl)benzoic acid (Example 5-1b) (450, mg,1.4 mmol), benzyl bromide (770 mg, 4.5 mmol), and cesium carbonate (1.5g, 4.5 mmol) in DMF (10 mL) were stirred at 80° C. for 2 hours. Thesolution was cooled to room temperature, diluted with H₂O (200 mL) andextract with ethyl acetate (3×, 100 mL). The combined organic layerswere dried over sodium sulfate, filtered and concentrated under vacuum.The residue was purified by silica gel chromotography (10% ethyl acetatein hexanes) to afford benzyl4-(N-benzyl-N-(4-methoxybenzyl)sulfamoyl)benzoate (517 mg, 73%) as awhite solid. ¹H NMR (400 MHz, CDCl₃) δ 3.76 (s, 3H), 4.28 (s, 2H), 4.32(s, 2H0, 5.40 (s, 2H), 6.79 (d, J=8 Hz, 2H), 6.96 (d, J=8 Hz, 2H),7.04-7.07 (m, 2H), 7.21-7.23 (m, 3H), 7.35-7.47 (m, 5H), 7.85 (d, J=8Hz, 2H), 8.17 (d, J=8.4 Hz, 2H).

Example 5-1b 4-(N-(4-methoxybenzyl)sulfamoyl)benzoic acid

4-(Chlorosulfonyl)benzoic acid (5 g, 22.7 mmol) was added, in threeportions as a solid, to a stirring solution of 4-methoxy benzylamine(6.1 g, 45 mmol) and triethylamine (2.3 g, 22.7 mmol) in acetone (100mL) cooled to 0° C. via an ice water bath over a 10 minute period. Theice bath was removed and the reaction was stirred for an additional 4hours. The reaction mixture was diluted with a solution of 5% aceticacid in H₂O (150 mL) and extracted with ethyl acetate (3×, 100 mL). Thecombined organic extracts were dried over sodium sulfate, filtered, andconcentrated on the rotovap. The resulting white solid was trituratedwith hexanes/ethyl acetate (9/1) to afford4-(N-(4-methoxybenzyl)sulfamoyl)benzoic acid (5.1 g, 70%) as a whitesolid. ¹H NMR (400 MHz, DMSO d₆) δ 3.68 (s, 3H), 3.91 (s, 2H), 6.79 (d,J=8.4 Hz, 2H), 7.10 (d, J=8.8 Hz, 2H), 7.80 (d, J=8.8 Hz, 2H), 8.02 (d,J=8.4 Hz, 2H).

Example 5-2 4-(N-(furan-2-ylmethyl)-N-(4-methoxybenzyl)sulfamoyl)benzoicacid

4-Methoxybenzyl4-(N-(furan-2-ylmethyl)-N-(4-methoxybenzyl)sulfamoyl)benzoate (Example5-2a) (750 mg, 1.4 mmol) was stirred a 2/2/1 mixture of aqueous 2NLiOH/THF/MeOH (45 mL) at ambient temperature for 3 hours. The solutionwas acidified with aqueous 1N aq HCl to a pH of ˜3 (ca. 100 mL) andextracted with ethyl acetate (3×, 100 mL). The combined organic extractswere dried over sodium sulfate and concentrated on the rotovap. Theresidue was taken up in MeOH (9 mL) and purified by reverse phased HPLC(5-95% ACN in H₂O gradient: 40 minutes) in three-3 mL aliquots. The purefractions were combined and concentrated to a white solid. The productwas dissolved in absolute ethanol and evaporated (4×, 20 mL) to providepure 4-(N-(furan-2-ylmethyl)-N-(4-methoxybenzyl)sulfamoyl)benzoic acid(205 mg, 36%) as a white solid. M.p. 151-152° C. ¹H NMR (DMSO-d₆, 400MHz):

3.71 (s, 3H), 4.24 (s, 2H), 4.27 (s, 2H), 6.14 (d, 1H, J=3.2 Hz), 6.26(m, 1H), 6.87 (d, 2H, J=9.2 Hz), 7.14 (d, 2H, J=8.8 Hz), 7.41 (s, 1H),7.89 (d, 2H, J=8 Hz), 8.06 (d, 2H, J=8.4 Hz), 13.48 (bs, 1H). MS 400(M−H).

The compound had an IC₅₀ on hT2R14 bitter receptor of 0.59 μM

Example 5-2a 4-methoxybenzyl4-(N-(furan-2-ylmethyl)-N-(4-methoxybenzyl)sulfamoyl)benzoate

4-(N-(furan-2-ylmethyl)sulfamoyl)benzoic acid (Example 5-2b) (500 mg,1.8 mmol), p-methoxy benzyl chloride (624 mg, 4.0 mmol), and cesiumcarbonate (1.3 g, 4.0 mmol) were dissolved in DMF (10 mL) and stirred at80° C. for 1 hour. The mixture was cooled to ambient temperature,diluted with H₂O (200 mL), and extracted with ethyl acetate (3×100 mL).The combined organics were dried over sodium sulfate and concentrated onthe rotovap. The product was purified by silica gel chromatography (15%ethyl acetate in hexanes) to provide the 4-methoxybenzyl4-(N-(furan-2-ylmethyl)-N-(4-methoxybenzyl)sulfamoyl)benzoate (753 mg,80%) as a clear oil. ¹H NMR (DMSO-d₆, 400 MHz): δ 3.70 (s, 3H), 3.75 (s,3H), 4.22 (s, 2H), 4.26 (s, 2H), 5.39 (s, 2H), 6.14 (d, 1H, J=3.2 Hz),6.25 (m, 1H), 6.87 (d, 2H, J=8.8 Hz), 6.97 (d, 2H, J=8.8 Hz), 7.13 (d,2H, J=8.4 Hz), 7.39 (m, 1H), 7.43 (d, 2H, J=8.4 Hz), 7.91 (d, 2H, J=8.4Hz), 8.07 (d, 2H, J=8.4 Hz).

Example 5-2b 4-(N-(furan-2-ylmethyl)sulfamoyl)benzoic acid

4-(Chlorosulfonyl)benzoic acid (5.0 g, 22.7 mmol) was added in threeportions over a 10 minute period to a stirring solution of furfurylamine (6.6 g, 68 mmol) in acetone (200 mL) cooled to 0° C. via an icewater bath. After addition of the sulfonyl chloride was complete the icebath was removed and the solution was stirred for 1 hour at ambienttemperature. The mixture was concentrated and subjected to silica gelchromatography (90% ethyl acetate, 8% hexanes and 2% acetic acid) toafford 4.4 of 4-(N-(furan-2-ylmethyl)sulfamoyl)benzoic acid (4.4 g, 68%)as a white solid. ¹H NMR (DMSO-d₆, 400 MHz):

4.04 (d, 2H, J=6 Hz), 6.13 (d, 1H, J=3.2 Hz), 6.25 (m, 1H), 7.43 (m,1H), 7.83 (d, 2H, J=8.4 Hz), 8.05 (d, 2H, J=8.4 Hz), 8.36 (t, 1H, J=6Hz), 13.4 (bs, 1H).

Example 5-3 4-(N-(4-ethoxybenzyl)-N-(furan-2-ylmethyl)sulfamoyl)benzoicacid

4-cyano-N-(4-ethoxybenzyl)-N-(furan-2-ylmethyl)benzenesulfonamide(Example 5-3a) (300 mg, 0.8 mmol) was stirred in a 1/1 mixture ofdioxane/1.5 N aqueous NaOH (100 mL) at 80° C. for 16 hours. The mixturewas cooled, acidified with 1N aqueous HCl (100 mL), and extracted withethyl acetate (3×, 75 mL). The combined organic extracts were dried oversodium sulfate, filtered, and concentrated on the rotovap. The solid wastriturated with ethyl acetate/hexanes (˜1/9) and collected by filtrationto afford 4-(N-(4-ethoxybenzyl)-N-(furan-2-ylmethyl)sulfamoyl)benzoicacid (250 mg, 69%) as a white solid. NMR (DMSO-d₆, 400 MHz): δ 1.29 (t,J=6.8 Hz, 3H), 3.97 (q, J=6.4 Hz, 2H), 4.23 (s, 2H), 4.27 (s, 2H), 6.15(d, J=3.2 Hz, 1H), 6.27 (m, 1H), 6.84 (d, J=8.8 Hz, 2H), 7.11 (d, J=8.8Hz, 2H), 7.39 (m, 1H), 7.87 (d, J=8.4 Hz, 2H), 8.00 (d, J=8.4 Hz, 2H).

The compound had an IC₅₀ on hT2R14 bitter receptor of 3.0 μM

Example 5-3a4-cyano-N-(4-ethoxybenzyl)-N-(furan-2-ylmethyl)benzenesulfonamide

4-cyanobenzene-1-sulfonyl chloride (600 mg, 2.9 mmol) was added to astirring solution of N-(4-ethoxybenzyl)-1-(furan-2-yl)methanamine(Example 5-3b) (685 mg, 2.9 mmol) and triethlyamine (455 mg, 4.5 mmol)in DCM (100 mL) and the reaction was stirred for 2 hours. The reactionwas diluted with H₂O (200 mL) and extracted with DCM (3×, 75 mL). Thecombined organic extracts were dried over sodium sulfate, filtered andconcentrated on the rotovap. The residue was purified by silica gelchromatography (10% ethyl acetate in hexanes) to afford4-cyano-N-(4-ethoxybenzyl)-N-(furan-2-ylmethyl)benzenesulfonamide (665mg, 68%) as an off white solid. NMR (DMSO-d₆, 400 MHz): δ 1.29 (t, J=7.2Hz, 3H), 3.97 (q, J=6.8 Hz, 2H), 4.23 (s, 2H), 4.27 (s, 2H), 6.15 (d,J=3.2 Hz, 1H), 6.27 (m, 1H), 6.84 (d, J=8.8 Hz, 2H), 7.11 (d, J=8.8 Hz,2H), 7.39 (m, 1H), 7.92 (d, J=8.4 Hz, 2H), 8.03 (d, J=8.4 Hz, 2H).

Example 5-3b 4N-(4-ethoxybenzyl)-1-(furan-2-yl)methanamine

4-Ethoxy benzaldehyde (5 g, 33 mmol) and furfuryl amine (4.2 g, 43 mmoL)in a mixture of methanol (50 mL), trimethylorthoformate (10 mL) and AcOH(1 mL) were stirred at room temperature, under an atmosphere of nitrogenfor 16 hours. Sodium borohydride (1.4 g, 35 mmol) was added, in 4portions, over a period of 30 minutes (exothermic reaction). Thereaction was stirred for an additional 2 hours at room temperature. Thesolvent was removed under vacuum and the residue taken up in ethylacetate (150 mL). The organic phase was washed with H₂O (200 mL) andaqueous phase was back extracted with ethyl acetate (2×, 100 mL). Thecombined organic layers were concentrated and the residue was purifiedon silica gel (70% ethyl acetate in hexanes with ˜0.5% triethylamine) toafford N-(4-ethoxybenzyl)-1-(furan-2-yl)methanamine (6.1 g, 80%) as aclear oil. NMR (CDCl₃, 400 MHz): δ 1.40 (t, J=7.2 Hz, 3H), 3.71 (s, 2H),3.76 (s, 2H), 4.02 (q, J=7.2 Hz, 2H), 6.17 (d, J=4 Hz, 1H), 6.31 (m,1H), 6.84 (d, J=8.8 Hz, 2H), 7.23 (d, J=8.8 Hz, 2H), 7.36 (m, 1H).

Example 5-4 4-(N-ethyl-N-(4-methoxybenzyl)sulfamoyl)benzoic acid

4-(N-(4-methoxybenzyl)sulfamoyl)benzoic acid (Example 5-1b) (160 mg, 0.5mmol) and Cesium Carbonate (325 mg, 1 mmol) were placed in a microwavevial and dissolved in 2 mL DMF. Ethyl iodide (155 mg, 1 mmol) was addedinto the reaction mixture. The reaction was placed in a microwavereactor and heated at 165° C. for 5 minutes. The reaction mixture wasdissolved in Ethyl Acetate and washed with water. The organic layer wasdried over sodium sulfate and evaporated under vacuum. The crude productwas dissolved in 4/1 solution of 6N NaOH(aq)/tetrahydrofuran (3 mL) andstirred at ambient temperature for 6 hours. The solution was acidifiedwith 3N HCl (aq) to a pH of ˜3 and the product was extracted with ethylacetate (3×, 75 mL). The combined organic extracts were dried oversodium sulfate and concentrated under vacuum. The residue was taken upin methanol (3 mL) and purified by reverse phased HPLC (5-95%acetonitrile in H₂O gradient: 25 minutes). The compound was known toinhibit the hT2R14 with IC₅₀ of 20 μM. Yield 35%. MS M+H calculated350.11, found 350.0.

The compound had an IC₅₀ on hT2R14 bitter receptor of 10 μM.

Example 5-5 4-(N-benzyl-N-(furan-2-ylmethyl)sulfamoyl)benzoic acid

4-(N-(furan-2-ylmethyl)sulfamoyl)benzoic acid (Example 5-2b) (140 mg,0.5 mmol) and Cesium Carbonate (325 mg, 1 mmol) were placed in amicrowave vial and dissolved in 2 mL DMF. (Bromomethyl)benzene (170 mg,1 mmol) was added into the reaction mixture. The reaction was placed ina microwave reactor and heated at 165° C. for 5 minutes. The reactionmixture was dissolved in Ethyl Acetate and washed with water. Theorganic layer was dried over sodium sulfate and evaporated under vacuum.The crude product was dissolved in 4/1 solution of 6NNaOH(aq)/tetrahydrofuran (3 mL) and stirred at ambient temperature for 6hours. The solution was acidified with 3N HCl (aq) to a pH of ˜3 and theproduct was extracted with ethyl acetate (3×, 75 mL). The combinedorganic extracts were dried over sodium sulfate and concentrated undervacuum. The residue was taken up in methanol (3 mL) and purified byreverse phased HPLC (5-95% acetonitrile in H₂O gradient: 25 minutes).Yield 35%. MS M+H calculated 372.4, found 372.0.

The compound had an IC₅₀ on hT2R14 bitter receptor of 4.6 μM

Example 5-6 4-(N-(furan-2-ylmethyl)-N-(3-methoxybenzyl)sulfamoyl)benzoicacid

Prepared as in Example 5-5 from 1-(bromomethyl)-3-methoxybenzene and4-(N-(furan-2-ylmethyl)sulfamoyl)benzoic acid (Example 5-2b). Yield 35%.MS M+H calculated 402.3, found 402.0.

The compound had an IC₅₀ on hT2R14 bitter receptor of 10 μM

Example 5-7 4-(N-(furan-2-ylmethyl)-N-(2-methoxybenzyl)sulfamoyl)benzoicacid

Prepared as in Example 5-5 from 1-(bromomethyl)-2-methoxybenzene and4-(N-(furan-2-ylmethyl)sulfamoyl)benzoic acid (Example 5-2b). Yield 35%.MS M+H calculated 402.3, found 402.0.

The compound had an IC₅₀ on hT2R14 bitter receptor of 12 μM

Example 5-8 4-(N-(4-propoxybenzyl)-N-(furan-2-ylmethyl)sulfamoyl)benzoicacid

4-cyano-N-(4-propoxybenzyl)-N-(furan-2-ylmethyl)benzenesulfonamide(Example 5-8a) (300 mg, 0.8 mmol) was stirred in a 1/1 mixture ofdioxane/1.5 N aqueous NaOH (100 mL) at 80° C. for 16 hours. The mixturewas cooled, acidified with 1N aqueous HCl (100 mL), and extracted withethyl acetate (3×, 75 mL). The combined organic extracts were dried oversodium sulfate, filtered, and concentrated on the rotovap. The solid wastriturated with ethyl acetate/hexanes (˜1/9) and collected by filtrationto afford 4-(N-(4-propoxybenzyl)-N-(furan-2-ylmethyl)sulfamoyl)benzoicacid (165 mg, 63%) as a white solid. NMR (DMSO-d₆, 400 MHz): δ 0.94 (t,J=7.6 Hz, 3H), 1.70 (m, J=6.8 Hz, 2H), 3.87 (t, J=6.4 Hz, 2H), 4.23 (s,2H), 4.27 (s, 2H), 6.13 (d, J=2.8 Hz, 1H), 6.27 (m, 1H), 6.84 (d, J=6.8Hz, 2H), 7.11 (d, J=8.8 Hz, 2H), 7.39 (m, 1H), 7.87 (d, J=6.8 Hz, 2H),8.05 (d, J=6.8 Hz, 2H), 13.45 (bs, 1H).

The compound had an IC₅₀ on hT2R14 bitter receptor of 2.5 μM

Example 5-8a4-cyano-N-(4-propoxybenzyl)-N-(furan-2-ylmethyl)benzenesulfonamide

4-cyanobenzene-1-sulfonyl chloride (600 mg, 2.9 mmol) was added to astirring solution of N-(4-propoxybenzyl)-1-(furan-2-yl)methanamine(Example 5-8b) (685 mg, 2.9 mmol) and triethlyamine (455 mg, 4.5 mmol)in DCM (100 mL) and the reaction was stirred for 2 hours. The reactionwas diluted with H₂O (200 mL) and extracted with DCM (3×, 75 mL). Thecombined organic extracts were dried over sodium sulfate, filtered andconcentrated on the rotovap. The residue was purified by silica gelchromatography (10% ethyl acetate in hexanes) to afford4-cyano-N-(4-propoxybenzyl)-N-(furan-2-ylmethyl)benzenesulfonamide (500mg, 50%) as an off white solid. NMR (DMSO-d₆, 400 MHz): δ 0.95 (t, J=7.2Hz, 3H), 1.70 (m, J=6.4 Hz, 2H), 3.88 (t, J=6.4 Hz, 2H), 4.25 (s, 2H),4.28 (s, 2H), 6.15 (d, J=3.2 Hz, 1H), 6.27 (m, 1H), 6.84 (d, J=6.8 Hz,2H), 7.11 (d, J=8.8 Hz, 2H), 7.39 (m, 1H), 7.93 (d, J=6.4 Hz, 2H), 8.01(d, J=6.4 Hz, 2H).

Example 5-8b 4N-(4-propoxybenzyl)-1-(furan-2-yl)methanamine

4-Propoxy benzaldehyde (5 g, 31 mmol) and furfuryl amine (3.9 g, 40mmol) in a mixture of methanol (50 mL), trimethylorthoformate (10 mL)and AcOH (˜1 mL) were stirred at room temperature, under an atmosphereof nitrogen for 16 hours. Sodium borohydride (1.4 g, 35 mmol) was addedin 4 portions, over a period of 30 minutes (exothermic reaction). Thereaction was stirred for an additional 2 hours at room temperature. Thesolvent was removed on the rotovap and the residue taken up in ethylacetate (150 mL). The organic phase was washed with H₂O (200 mL) andaqueous phase was back extracted with ethyl acetate (2×, 100 mL). Thecombined organic layers were concentrated and the residue was purifiedon silica gel (70% ethyl acetate in hexanes with ˜2% triethylamine) toafford N-(4-propoxybenzyl)-1-(furan-2-yl)methanamine (5.3 g, 75%) as ayellow oil. NMR (CDCl₃, 400 MHz): δ 1.03 (t, J=7.2 Hz, 3H), 1.79 (m,J=6.4 Hz, 2H), 3.71 (s, 2H), 3.76 (s, 2H), 3.90 (t, J=6.8 Hz, 2H), 6.17(d, J=3.2 Hz, 1H), 6.32 (m, 1H), 6.85 (d, J=8.4 Hz, 2H), 7.22 (d, J=8.8Hz, 2H), 7.37 (m, 1H).

Additional compounds were experimentally tested and found to have arelatively high level of effectiveness as inhibitors of hT2R14 bitterreceptor. The results of that testing are shown below in Table B.

TABLE B Compound hT2R14 No. Compound IC₅₀ (μM) 5-9

4-(N,N-diisobutylsulfamoyl)benzoic acid 15

Example 5-10 4-(N-(4-fluorobenzyl)-N-(4-methoxybenzyl)sulfamoyl)benzoicacid

Methyl 4-(N-(4-fluorobenzyl)-N-(4-methoxybenzyl)sulfamoyl)benzoate(Example 5-10a) (3.7 g, 8.3 mmol) was dissolved in MeOH/THF (1:1.5, 30mL) and treated with aqueous NaOH (3N, 15 mL). The mixture was stirredat ambient temperature overnight, then MeOH and THF were removed invacuo. The resulting aqueous solution was acidified with 6 N aq HCl to apH of ˜3 and extracted with EtOAc (3×40 mL). The combined organic layerswere washed with water and brine, dried over Na₂SO₄ and concentrated.The crude product was purified by recrystallization from EtOH to affordpure 4-(N-(4-fluorobenzyl)-N-(4-methoxybenzyl)sulfamoyl)benzoic acid asa white crystalline solid (2.1 g, 58.6%). MS (M−H, 428.1); ¹H NMR (400MHz, DMSO-d6): δ, ppm: 3.66 (s, 3H), 4.23 (s, 2H), 4.26 (s, 2H), 6.72(d, 2H, J=8 Hz), 6.95 (m, 6H), 7.92 (d, 2H J=8 Hz), 8.07 (d, 2H, J=8Hz). The compound had an IC₅₀ on hT2R14 bitter receptor of 1.97 μM.

Example 5-10a Methyl4-(N-(4-fluorobenzyl)-N-(4-methoxybenzyl)sulfamoyl)-benzoate

Methyl 4-(N-(4-methoxybenzyl)sulfamoyl)benzoate (Example 5-10b) (4.3 g,12.8 mmol) was dissolved in acetone (70 mL). Cesium carbonate (8.57 g,25.6 mmol) and (4-fluorobenzyl bromide (1.76 mL, 14.08 mmol) were addedand the mixture was stirred at room temperature overnight. The inorganicsalts were filtered off and acetone was removed in vacuo. The residuewas re-dissolved in ethyl acetate, washed with water and brine, then theorganic layer was dried over magnesium sulfate and concentrated. Thecrude product was purified by re-crystallization with ethylacetate/hexanes to afford pure Methyl4-(N-(4-fluorobenzyl)-N-(4-methoxybenzyl)sulfamoyl)benzoate (3.7 g, 65%)as a white solid. ¹H NMR (400 MHz, CDCl₃): δ, ppm: 3.77 (s, 3H), 3.98(s, 3H), 4.27 (s, 2H), 4.28 (s, 2H), 6.74 (d, 2H, J=8 Hz), 6.92 (m, 4H),7.03 (m, 2H), 7.88 (d, 2H J=8 Hz), 8.16 (d, 2H, J=8 Hz).

Example 5-10b Methyl 4-(N-(4-methoxybenzyl)sulfamoyl)benzoate

To a solution of methyl 4-(chlorosulfonyl)benzoate (Example 5-10c) (4 g,17.09 mmol) in dichloromethane (40 mL) at 0° C. in an ice bath, wasadded (4-methoxyphenyl)methanamine (2.56 mL, 19.65 mmol) andtriethylamine (2.38 mL, 17.1 mmol). The ice bath was then removed andthe mixture was allowed to warm to ambient temperature with stirring foran additional 2 hours. Upon completion (monitored by TLC 40% ethylacetate/hexanes), the solvent was removed in vacuo. The residue wasre-dissolved in ethyl acetate (200 mL), washed with 1N HCl (aq, 20 mL),water (20 mL) and brine (20 mL) then dried over magnesium sulfate. Thesolution was concentrated and the product purified by re-crystallizationwith hot ethyl acetate/hexanes to afford pure methyl4-(N-(4-methoxybenzyl)sulfamoyl)benzoate (4.3 g, 74.8%) as a whitesolid. ¹H NMR (400 MHz, DMSO-d6): δ, ppm: 3.67 (s, 3H), 3.78 (s, 3H),3.93 (s, 2H), 6.78 (m, 2H), 7.09 (m, 2H), 7.87 (m, 2H), 8.08 (m, 2H),8.25 (br, s, 1H).

Example 5-10c Methyl 4-(chlorosulfonyl)benzoate

4-Chlorosulfonyl benzoic acid (5 g, 23 mmol) and thionyl chloride (20mL) in dichloroethane (10 mL) was heated to 80° C. for 2 hr. Thereaction mixture was concentrated via rotary evaporation to give abrownish solid. The solid was chilled on ice for 5 minutes and ice-coldmethanol (40 mL) was added with stirring at 0° C. for 5 minutes. Thereaction mixture was allowed to warm to ambient temperature and stirredan additional 10 min. The addition of ice-cold water (40 mL), produced awhite solid that was collected by filtration and dried under vacuum toafford pure methyl 4-(chlorosulfonyl)benzoate (4.5 g, 84%). ¹H NMR (400MHz, DMSO-d6): δ, ppm: 3.84 (s, 3H), 7.70 (d, 2H, J=8.4 Hz), 7.93 (d,2H, J=8.4 Hz).

Example 5-114-((N-benzyl-4-methylphenylsulfonamido)methyl)cyclohexane-carboxylicacid

To the suspension of 4-(aminomethyl)cyclohexanecarboxylic acid (1.57 g,10 mmol) in 100 ml of 2,2-dimethoxypropane, was added HCl (10 ml, 36%aq). The mixture was stirred at ambient temperature for 18 h and thenconcentrated. The residue was dissolved in a minimum volume of MeOH andDiethyl ether was added to precipitate the HCl salt, of methyl4-(aminomethyl)cyclohexanecarboxylate as an off-white solid. Thismaterial was used without further purification or characterization.

To the mixture of the HCl salt of methyl4-(aminomethyl)cyclohexanecarboxylate (208 mg, 1 mmol) in 5 mL ofdichloromethane, at 0° C. in an ice bath, were added triethylamine (360uL, 2.58 mmol) and 4-methylbenzene-1-sulfonyl chloride (190 mg, 1 mmol).The ice bath was allowed to warm slowly to ambient temperature andstirred overnight. The solvent was removed in vacuo. The residue wasre-dissolved in ethyl acetate (20 mL), washed with 1N HCl (5 mL), water(5 mL) and brine (5 mL), then dried over magnesium sulfate andconcentrated. This crude product (162 mg, 0.5 mmol) was redissolved inacetone (5 mL) and treated with potassium carbonate (110 mg, 0.79 mmol)and (4-fluorophenyl)methanamine (1.76 mL, 14.08 mmol). The mixture wasstirred in a pressure vessel at 80° C. overnight, then cooled and theinorganic salts were filtered off. Acetone was removed in vacuo and theresidue was re-dissolved in ethyl acetate and washed with water followedby brine. The organic layer was dried over magnesium sulfate andconcentrated. The crude product (162 mg, 0.4 mmol) was dissolved inMeOH/THF (1:1.5, 10 mL) and treated with aqueous NaOH (190N, 400 uL).The mixture was stirred at 100° C. for 20 min in a microwave, and thenMeOH and THF were removed in vacuo. The residue was acidified with 6 Naq HCl to a pH of ˜3 and extracted with EtOAc; the combined organiclayers were washed with water and brine, dried over Na₂SO₄ andconcentrated. The crude product was purified by recrystallization fromEtOH to afford pure4-((N-benzyl-4-methylphenylsulfonamido)methyl)cyclohexanecarboxylic acidas a white solid (120 mg, 74%). MS (M+H, 402); ¹H NMR (400 MHz,DMSO-d6): δ, ppm: 0.68 (m, 2H)), 0.91 (m, 2H), 1.06 (br, s, 1H), 1.50(d, 2H), 1.72 (d, 2H), 2.0 (1H), 2.41 (s, 3H), 2.86 (m, 2H), 4.21 (s,2H), 7.30 (m, 5H), 7.43 (m, 2H), 7.73 (m, 2H), 11.97 (br, s, 1H).

The compound had an IC₅₀ on hT2R14 bitter receptor of 0.014 μM.

Example 5-12 4-(N-(3-fluorobenzyl)-N-(4-methoxybenzyl)sulfamoyl)benzoicacid

Methyl 4-(N-(4-methoxybenzyl)sulfamoyl)benzoate (Example 5-10b) (500 mg,1.49 mmol), 3-fluorobenzyl bromide (280 mg, 2.98 mmol), and cesiumcarbonate (971 mg, 2.98 mmol) were placed in DMF (12 mL) and stirred at90° C. for 4 hours. The solution was cooled to ambient temperature,diluted with H₂O (200 mL) and extracted with ethyl acetate (3×, 100 mL).The combined organic layers were dried over MgSO₄, filtered andconcentrated under vacuum. The residue was purified by silica gelchromatography (10-20% ethyl acetate in hexane) to afford methyl4-(N-(3-fluorobenzyl)-N-(4-methoxybenzyl)sulfamoyl)benzoate (528 mg,80%) as a white solid.

Methyl 4-(N-(3-fluorobenzyl)-N-(4-methoxybenzyl)sulfamoyl)benzoate (500mg, 1.12 mmol) was dissolved in MeOH/THF (1:1, 40 mL) and treated with asolution of aqueous NaOH (10 N, 8 ml). The mixture was stirred atambient temperature overnight, then MeOH and THF were removed by rotaryevaporation. The resulting aqueous solution was washed with EtOAc (10mL) and acidified with 6 N aq HCl (˜15 mL) to pH˜4. The aqueous solutionwas extracted with EtOAc (3×, 40 mL) and the combined organic layerswere washed with water, brine, dried over MgSO₄ and concentrated. Thecrude product was purified by recrystallization from EtOH to afford thetitle compound4-(N-(3-fluorobenzyl)-N-(4-methoxybenzyl)sulfamoyl)benzoic acid as awhite crystalline solid (150 mg) in 30% yield.

MS (M−H, 428.1); ¹H NMR (400 MHz, DMSO-d6): δ, ppm: 3.65 (s, 3H), 4.27(s, 2H), 4.29 (s, 2H), 6.75-7.00 (m, 7H), 7.20 (m, 1H), 7.95 (d, 2H, J=8Hz); 8.10 (d, 2H, J=8 Hz).

Example 5-13 4-(N-benzyl-N-(2,4-dimethoxybenzyl)sulfamoyl)benzoic acid

Prepared as in example 5-10 from (2,4-dimethoxyphenyl)methanamine,methyl 4-(chlorosulfonyl)benzoate (Example 5-10c) and benzyl bromide. MS(M−H, 440.10); ¹H NMR (400 MHz, DMSO-d6): δ, ppm: 3.50 (s, 63H), 3.66(s, 3H), 4.20 (s, 2H), 4.34 (s, 2H), 6.29 (s, 1H), 6.33 (d, J=8.0 Hz,1H), 6.91 (d, J=8.8 Hz, 1H), 7.12-7.23 (m, 5H), 7.80 (d, J=8.0 Hz, 2H),8.01 (d, J=8.4 Hz, 2H), 13.49 (s, 1H).

Example 5-14 4-(N-(3-chlorobenzyl)-N-(4-methoxybenzyl)sulfamoyl)benzoicacid

Prepared as in example 5-10 from Methyl4-(N-(4-methoxybenzyl)sulfamoyl)benzoate (Example 5-10b) and1-(bromomethyl)-3-chlorobenzene. MS (M−H, 444.1); ¹H NMR (400 MHz,DMSO-d6): δ, ppm: 3.69 (s, 3H), 4.30 (m, 4H), 6.76-7.24 (m, 8H), 7.99(m, 2H), 8.13 (m, 2H).

The compound had an IC₅₀ on hT2R14 bitter receptor of 1.88 μM.

Example 5-15 4-(N-benzyl-N-(2,4,6-trimethoxybenzyl)sulfamoyl)benzoicacid

Prepared as in example 5-10 from (2,4,6-trimethoxyphenyl)methanamine,methyl 4-(chlorosulfonyl)benzoate (Example 5-10c) and benzyl bromide. MS(M−H, 470.10); ¹H NMR (400 MHz, DMSO-d6): δ, ppm: 3.45 (s, 6H), 3.69 (s,3H), 4.26 (s, 2H), 4.28 (s, 2H), 5.98 (s, 2H), 7.11-7.26 (m, 5H), 7.82(d, J=8.0 Hz, 2H), 8.07 (d, J=8 Hz, 2H), 13.49 (s, 1H). Elementalanalysis (found, %): C, 61.05; H, 5.49; N, 2.98; (calculated, %): C,61.13; H, 5.34 and N, 2.97

The compound had an IC₅₀ on hT2R14 bitter receptor of 10.76 μM.

Example 5-16 4-((N-benzyl-N-(4-methoxybenzyl)sulfamoyl)methyl)benzoicacid

Prepared as in example 5-10 from Methyl4-(N-(4-methoxybenzyl)sulfamoyl)benzoate (Example 5-10b) and benzylbromide. MS (M−H, 424.1); ¹H NMR (400 MHz, DMSO-d6): δ, ppm: 3.72 (s,3H), 4.18 (d, 2H), 4.55 (s, 2H), 6.83 (m, 2H), 7.12 (m, 2H), 7.21 (m,2H), 7.28 (m, 3H), 7.43 (m, 2H), 7.93 (m, 2H).

Example 5-17 4-(N-(4-methoxybenzyl)-N-propylsulfamoyl)benzoic acid

Prepared as in example 5-10 from Methyl4-(N-(4-methoxybenzyl)sulfamoyl)benzoate (Example 5-10b) and n-propylbromide. MS (M−H, 362.1); ¹H NMR (400 MHz, CDCl3): δ, ppm: 0.70 (m, 3H),1.35 (m, 3H), 3.08 (m, 2H), 3.73 (s, 3H), 4.31 (s, 2H), 6.83 (m, 2H),7.17 (m, 2H), 7.93 (m, 2H), 8.23 (m, 2H).

The compound had an IC₅₀ on hT2R14 bitter receptor of 3.75 μM.

Example 5-18 4-(N-(4-methoxybenzyl)-N-phenylsulfamoyl)benzoic acid

Prepared as in example 5-10 from aniline, methyl4-(chlorosulfonyl)benzoate (Example 5-10c) and1-(chloromethyl)-4-methoxybenzene. MS (M−H, 396.1); ¹H NMR (400 MHz,DMSO-d6): δ, ppm: 3.66 (s, 3H), 4.73 (s, 2H), 6.78 (d, J=8.4 Hz, 2H),7.00 (d, J=7.6 Hz, 2H), 7.12 (d, J=8.0 Hz, 2H), 7.24 (d, J=8.0 Hz, 2H),8.11 (d, J=8.4 Hz, 2H), 13.49 (s, 1H).

Example 5-19 4-(N-(4-methoxybenzyl)-N-phenethylsulfamoyl)benzoic acid

Prepared as in example 5-10 from 2-phenylethanamine, methyl4-(chlorosulfonyl)-benzoate (Example 5-10c) and1-(chloromethyl)-4-methoxybenzene. MS (M−H, 424.1); ¹H NMR (400 MHz,DMSO-d6): δ, ppm: 2.51 (m, 2H), 3.23 (m, 2H), 3.74 (s, 3H), 4.31 (s,2H), 4.31 (s, 2H), 6.92 (m, 2H), 6.98 (m, 2H), 7.20-7.27 (m, 5H), 7.85(m, 2H), 8.06 (m, 2H).

The compound had an IC₅₀ on hT2R14 bitter receptor of 4.58 μM.

Example 5-204-((N-(4-fluorobenzyl)-4-methylphenylsulfonamido)methyl)-cyclohexanecarboxylicacid

Prepared as in example 5-11 from 1-(bromomethyl)-4-fluorobenzene,4-(aminomethyl)cyclohexanecarboxylic acid and4-methylbenzene-1-sulfonylchloride. MS (M+H, 420); ¹H NMR (400 MHz,DMSO-d6): δ, ppm: 0.71 (m, 2H)), 0.95 (m, 2H), 1.15 (br, s, 1H), 1.51(d, 2H), 1.76 (d, 2H), 2.0 (1H), 2.40 (s, 3H), 2.85 (m, 2H), 4.22 (s,2H), 7.14 (m, 2H), 7.32 (m, 2H), 7.40 (m, 2H), 7.69 (m, 2H), 11.93 (br,s, 1H).

The compound had an IC₅₀ on hT2R14 bitter receptor of 0.083 μM.

Example 5-214-((4-acetamido-N-benzylphenylsulfonamido)methyl)-cyclohexanecarboxylicacid

Prepared as in example 5-11 from acetamidobenzene-1-sulfonyl chloride,4-(aminomethyl)cyclohexanecarboxylic acid and benzyl bromide. MS (M+H,445.2); ¹H NMR (400 MHz, DMSO-d6): δ, ppm: 0.65 (m, 2H), 0.68 (m, 2H),1.05 (m, 1H), 1.48 (m, 2H), 1.70 (m, 2H), 1.92 (m, 1H), 2.00 (s, 3H),2.82 (s, 2H), 4.21 (s, 2H), 7.27 (m, 5H), 7.76 (m, 4H), 7.62 (m, 2H),10.1 (s, 1H), 11.93 (br, s, 1H).

The compound had an IC₅₀ on hT2R14 bitter receptor of 1.619 μM.

Example 5-224-((N-(4-fluorobenzyl)phenylsulfonamido)methyl)-cyclohexanecarboxylicacid

Prepared as in example 5-11 from benzenesulfonyl chloride,4-(aminomethyl)-cyclohexanecarboxylic acid and1-(bromomethyl)-4-fluorobenzene. MS (M+H, 406); ¹H NMR (400 MHz,DMSO-d6): δ, ppm: 0.77 (m, 2H), 0.94 (m, 2H), 0.98 (m, 2H), 1.50 (m,2H0, 1.75 (m, 2H), 1.77 (m, 1H), 2.80 (d, 2H), 4.28 (s, 2H), 7.20 (m,2H), 7.38 (m, 2H), 7.62 (m, 2H), 7.70 (m, 1H), 7.88 (m, 2H), 11.93 (br,s, 1H).

The compound had an IC₅₀ on hT2R14 bitter receptor of 0.240 μM.

Example 5-234-(N-(cyclohexylmethyl)-N-(4-methoxybenzyl)sulfamoyl)-benzoic acid

Prepared as in example 5-10 from Methyl4-(N-(4-methoxybenzyl)sulfamoyl)benzoate (Example 5-10b) andcyclohexylmethanamine. MS (M−H, 416.1); NMR (400 MHz, DMSO-d6): δ, ppm:0.63 (m, 2H), 0.87 (m, 3H), 0.94 (m, 1H), 1.25-1.52 (m, 5H), 1.70 (m,2H), 2.86 (m, 2H), 3.70 (s, 3H), 4.22 (s, 2H), 6.84 (m, 2H), 7.16 (m,2H), 7.91 (m, 2H), 7.62 (m, 2H), 8.09 (m, 2H).

The compound had an IC₅₀ on hT2R14 bitter receptor of 3.47 μM.

Example 5-244-((N-(4-fluorobenzyl)-1-phenylmethylsulfonamido)methyl)-cyclohexanecarboxylicacid

Prepared as in example 5-10 from phenylmethanesulfonyl chloride andmethyl 4-(aminomethyl)cyclohexanecarboxylate and1-(bromomethyl)-4-fluorobenzene. MS (M−H, 418); ¹NMR (400 MHz, DMSO-d6):δ, ppm: 0.639 (m, 2H), 0.897 (m, 2H), 1.034 (m, 1H), 1.467 (d, broad,2H, J=11.2 Hz), 1.709 (d, broad, 2H, J=11.2 Hz), 1.961 (m, 1H), 2.828(d, 2H, J=7.6 Hz), 4.207 (s, 2H), 4.449 (s, 2H), 7.155 (t, 2H, J=9.2Hz), 7.377 (m, 7H), 12 (s, broad, 1H)

The compound had an IC₅₀ on hT2R14 bitter receptor of 9.57 μM.

Example 5-25 4-(N-(2-cyanobenzyl)-N-(4-methoxybenzyl)sulfamoyl)benzoicacid

Prepared as in example 5-10 from Methyl4-(N-(4-methoxybenzyl)sulfamoyl)-benzoate (Example 5-10b) andalpha-bromo-o-tolunitrile. MS (M−H, 435.1); ¹H NMR (400 MHz, DMSO-d6):δ, ppm: 3.664 (s, 2H), 4.348 (s, 2H), 4.498 (s, 2H), 6.728 (d, 2H, J=8.4Hz), 7.036 (d, 2H, j=8.4 Hz), 7.352 (t, 2H, J=9.2 Hz), 7.548 (t, 1H,J=7.6 Hz), 7.640 (d, 1H, J=7.6 Hz), 8.003 (d, 2H, J=8 Hz), 8.139 (d, 2H,J=8.4 Hz), 13.559 (s, broad, 1H).

The compound had an IC₅₀ on hT2R14 bitter receptor of 4.61 μM.

Example 5-264-(N-(4-acetamidobenzyl)-N-(4-methoxybenzyl)sulfamoyl)benzoic acid

Prepared as in example 5-10 from Methyl4-(N-(4-methoxybenzyl)sulfamoyl)benzoate (Example 5-10b) andN-(4-(chloromethyl)phenyl)acetamide. MS (M−H, 467.1); ¹H NMR (400 MHz,DMSO-d6): δ, ppm: 2.0 (s, 3H), 3.69s, 3H), 4.23 (s, 4H), 6.78 (d, 2H,J=7.6 Hz), 6.98 (m, 4H), 7.41 (d, 2H, J=8 Hz), 7.94 (d, 2H, J=8 Hz),8.09 (d, 2H, J=8 Hz), 9.90 (s, 1H).

Example 5-274-(N-(furan-3-ylmethyl)-N-(4-methoxybenzyl)sulfamoyl)benzoic acid

110 mg of 1-(furan-3-yl)-N-(4-methoxybenzyl)methanamine (example 5-27a)was mixed with methyl 4-(chlorosulfonyl)benzoate (example 5-10c) (117mg, 0.5 mmol) and triethyl amine (100 uL) in DCM (5 mL). The mixture wasstirred overnight at ambient temperature and concentrated. The residuewas re-dissolved in ethyl acetate (20 mL), washed with 1N HCl (aq., 2mL), followed by water (5 mL) and brine (5 mL) then dried over magnesiumsulfate. The crude product was purified by preparative TLC (40% EthylAcetate/hexanes) to give methyl4-(N-(furan-3-ylmethyl)-N-(4-methoxybenzyl)sulfamoyl)benzoate as a whitesolid. Saponification as in example 5-10 gave4-(N-(furan-3-ylmethyl)-N-(4-methoxybenzyl)sulfamoyl)benzoic acid as awhite crystalline solid (68 mg, 64% yield). MS (M−H, 400.1); ¹H NMR (400MHz, DMSO-d6): δ, ppm: 3.72 (s, 3H), 4.13 (s, 2H), 4.26 (s, 2H), 5.99(s, 1H), 6.87 (m, 4H), 6.87 (d, 2H, J=8.8 Hz), 7.13 (d, 2H, J=8.8 Hz),7.39 (s, 1H), 7.49 (s, 1H), 7.95 (d, 2H, J=8.4 Hz), 8.09 (d, 2H, J=8.8Hz).

Example 5-27a 1-(furan-3-yl)-N-(4-methoxybenzyl)methanamine

A mixture of 3-Furaldehyde (5 mmol, 437 μL) and(4-methoxyphenyl)methanamine in MeOH (20 mL) was stirred at ambienttemperature overnight then sodium borohydride (300 mg, 7.89 mmol) wasslowly added. The resulting mixture was stirred at room temperature for1.5 minutes and quenched with NaOH (1 N, aq). Methanol was removed invacuo and the resulting slurry was redissolved in ethyl acetate then,washed with water, brine, dried over sodium sulfate and concentrated.Purification by silica gel chromatography (Ethyl acetate: Hexanes 7:3)gave 1-(furan-3-yl)-N-(4-methoxybenzyl)methanamine as an oil. MS (M+H,218.10); ¹H NMR (400 MHz, CDCl3): δ, ppm: 3.64 (s, 2H), 3.74 (s, 2H),3.80 (s, 3H), 6.39 (m, 1H), 6.86 (m, 1H), 6.88 (m, 1H), 7.23 (m, 1H),7.25 (m, 1H), 7.35 (m, 1H), 7.38 (m, 1H).

Example 5-28 4-(N,N-dibenzylsulfamoyl)benzoic acid

Prepared as in example 5-27 from dibenzylamine and methyl4-(chlorosulfonyl)-benzoate (Example 5-10c). MS (M−H, 380.1); ¹H NMR(400 MHz, DMSO-d6): δ, ppm: 4.52 (s, 4H), 7.10 (m, 4H), 7.25 (m, 6H),8.00 (d, 2H, J=8.4 Hz), 8.15 (d, 2H J=8.4 Hz), 13.5 (s, broad, 1H).

The compound had an IC₅₀ on hT2R14 bitter receptor of 7.74 μM.

Example 5-29 4-(N-(4-methoxybenzyl)-N-methylsulfamoyl)benzoic acid

Prepared as in example 5-27 from 1-(4-methoxyphenyl)-N-methylmethanamineand methyl 4-(chlorosulfonyl)-benzoate (Example 5-10c). MS (M−H, 335.1);¹H NMR (400 MHz, DMSO-d6): δ, ppm: 2.51 (s, 3H), 3.71 (s, 3H), 4.05 (s,2H), 6.88 (d, 2H), 7.18 (d, 2H), 7.91 (d, 2H); 8.13 (d, 2H). Elementalanalysis: (found): C, 57.47%, H, 4.77% and N, 4.31%; (theoretical): C,57.30%, H, 5.11% and N, 4.18%

Example 5-30 4-(N,N-bis(4-methoxybenzyl)sulfamoyl)benzoic acid

Prepared as in example 5-27 from bis(4-methoxybenzyl)amine and methyl4-(chlorosulfonyl)-benzoate (Example 5-10c). MS (M−H, 440.1); ¹H NMR(400 MHz, DMSO-d6): δ, ppm: 3.68 (s, 6H), 4.20 (s, 4H), 6.77 (d, 4H,J=10 Hz), 6.98 (d, 4H, J=10 Hz), 7.92 (dd, 2H J=8 Hz), 8.06 (dd, 2H, J=8Hz). Elemental analysis: (found): C, 62.45%, H, 5.19% and N, 3.06%;(theoretical): C, 62.57%, H, 5.25% and N, 3.17%

The compound had an IC₅₀ on hT2R14 bitter receptor of 4.14 μM.

Example 5-31 4-(N-(2-fluorobenzyl)-N-(4-methoxybenzyl)sulfamoyl)benzoicacid

Prepared as in example 5-10 from Methyl4-(N-(4-methoxybenzyl)sulfamoyl)-benzoate (Example 5-10b) and1-(bromomethyl)-2-fluorobenzene. ¹H NMR (400 MHz, DMSO-d6): δ, ppm: 3.66(s, 3H), 4.29 (s, 2H), 4.37 (s, 2H), 6.72 (d, 2H, J=8 Hz), 7.01-7.03 (m,6H), 7.93 (d, 2H, J=8 Hz), 8.08 (d, 2H, J=8 Hz).

Example 5-324-(N-(2,5-difluorobenzyl)-N-(4-methoxybenzyl)sulfamoyl)-benzoic acid

Prepared as in example 5-10 from Methyl4-(N-(4-methoxybenzyl)sulfamoyl)-benzoate (Example 5-10b) and2-(bromomethyl)-1,4-difluorobenzene. ¹H NMR (400 MHz, DMSO-d6): δ, ppm:3.66 (s, 3H), 4.31 (s, 2H), 4.33 (s, 2H), 6.74-7.06 (m, 7H), 7.95 (d,2H, J=8 Hz), 8.09 (d, 2H, J=8 Hz).

Example 5-334-(N-(2,3-difluorobenzyl)-N-(4-methoxybenzyl)sulfamoyl)benzoic acid

Prepared as in example 5-10 from Methyl4-(N-(4-methoxybenzyl)sulfamoyl)-benzoate (Example 5-10b) and1-(bromomethyl)-2,3-difluorobenzene. ¹H NMR (400 MHz, DMSO-d6): δ, ppm:3.30 (s, 3H), 4.29 (s, 2H), 4.32 (s, 2H), 6.87 (d, 2H, J=8 Hz),7.02-7.20 (m, 5H), 7.95 (d, 2H, J=8 Hz). 8.05 (d, 2H, J=8 Hz).

Example 5-344-(N-(3-methoxybenzyl)-N-(4-methoxybenzyl)sulfamoyl)-benzoic acid

Prepared as in example 5-10 from Methyl4-(N-(4-methoxybenzyl)sulfamoyl)benzoate (Example 5-10b) and3-methoxybenzyl bromide. MS (M−H, 440.50); ¹H NMR (400 MHz, DMSO-d6): δ,ppm: 3.58 (s, 3H), 3.68 (s, 3H), 4.24 (s, 2H), 4.25 (s, 2H), 6.50 (s,1H), 6.64 (d, J=4 Hz, 1H), 6.73 (m, 1H), 6.77 (d, J=8 Hz, 2H), 7.00 (d,J=8 Hz, 2H), 7.12 (t, J=8 Hz, 1H), 7.94 (d, J=8 Hz, 2H), 8.09 (d, J=8Hz, 2H), 13.49 (s, 1H).

The compound had an IC₅₀ on hT2R14 bitter receptor of 2.46 μM.

Example 5-35 4-(N-benzyl-N-(4-methoxyphenyl)sulfamoyl)benzoic acid

Prepared as in example 5-10 from Methyl4-(N-(4-methoxyphenyl)sulfamoyl)-benzoate (example 5-35a) and benzylbromide MS (M−H, 396); ¹H NMR (400 MHz, DMSO-d6): δ, ppm: 3.66 (s, 3H),4.75 (s, 2H), 6.76 (d, J=8 Hz, 2H), 6.90 (d, J=8 Hz, 2H), 7.23 (m, 5H),7.74 (d, J=8 Hz, 2H), 8.11 (d, J=8 Hz, 2H), 13.51 (s, 1H).

Example 5-35a Methyl 4-(N-(4-methoxyphenyl)sulfamoyl)benzoate

To 4-methoxybenzenamine (580 mg, 4.71 mmol) and triethylamine (1.48 mL,10.7 mmol) in dichloromethane (10 mL) was added methyl4-(chlorosulfonyl)benzoate (1.00 g, 4.28 mmol). This mixture was stirredfor 16 hours at room temperature. The reaction was diluted withdichloromethane (50 mL) and washed consecutively with water, 10% citricacid, and brine. The organics were dried over sodium sulfate andconcentrated via rotovap. The resulting crude material waschromatographed on silica gel using 100% dichloromethane as eluentaffording Methyl 4-(N-(4-methoxyphenyl)sulfamoyl)benzoate as a whitecrystalline solid (400 mg, 30% yield)

Example 5-364-(N-(3,4-difluorobenzyl)-N-(4-methoxybenzyl)sulfamoyl)benzoic acid

Prepared as in example 5-10 fromN-(3,4-difluorobenzyl)(4-methoxyphenyl)-methanamine (example 5-36a) andmethyl 4-(chlorosulfonyl)benzoate (example 5-10c). MS (M−H, 446); ¹H NMR(400 MHz, DMSO-d6): δ ppm: 3.69 (s, 3H), 4.20 (s, 4H), 6.73 (d, J=8.8Hz, 2H), 6.93 (m, 2H), 6.98 (d, J=8.8 Hz, 2H), 7.22 (m, 1H), 7.74 (d,J=8.4 Hz, 2H), 7.99 (d, J=8.4 Hz, 2H).

Example 5-36a N-(3,4-difluorobenzyl)(4-methoxyphenyl)methanamine

To (4-methoxyphenyl)methanamine (1.77 mL, 13.6 mmol) and acetic acid(2.7 mL, 45 mmol) in dichloromethane (15 mL) was added3,4-difluorobenzaldehyde (1.0 m, 9.08 mmol). This mixture was heated inthe microwave at 100° C. for 15 min. The reaction was cooled to roomtemperature and macroporous cyanoborohydride resin (9.8 g, 22.7 mmol)was added in portions. This mixture was stirred at room temperature for16 hours. The resin was filtered off and rinsed with dichloromethane andthe organics were washed with saturated sodium bicarbonate untilbubbling ceased. The organics were dried over sodium sulfate andconcentrated via rotovap. The resulting crude material was purified bysilica gel chromatography using methanol dichloromethane gradient aseluent to afford N-(3,4-difluorobenzyl)(4-methoxyphenyl)methanamine as ayellowish oil (1.9 g, 80% yield). MS (M+H, 264); ¹H NMR (400 MHz,DMSO-d6): δ, ppm: 2.63 (br s, 1H), 3.56 (s, 2H), 3.61 (s, 2H), 3.71 (s,3H), 6.84 (d, J=8.8 Hz, 2H), 7.14 (m, 1H), 7.21 (d, J=8.4 Hz, 2H), 7.34(m, 2H).

Example 5-374-((N-(4-fluorobenzyl)-4-methylphenylsulfonamido)methyl)benzoic acid

Prepared as in example 5-11 from 4-(aminomethyl)phenylcarboxylic acid,4-methylbenzene-1-sulfonylchloride and 4-fluorobenzyl bromide. ¹H NMR(400 MHz, DMSO-d6): δ, ppm: 3.11 (s, 3H), 4.21 (s, 2H), 4.24 (s, 2H),6.94-7.08 (m, 6H), 7.40-7.42 (d, 2H, J=8 Hz), 7.63 (d, 2H, J=8 Hz). 7.73(d, 2H, J=8 Hz)

The compound had an IC₅₀ on hT2R14 bitter receptor of 0.054 μM.

Example 5-384-((4-carboxy-N-(4-methoxybenzyl)phenylsulfonamido)methyl)-benzoic acid

Prepared as in example 5-10 from methyl 4-(bromomethyl)benzoate andMethyl 4-(N-(4-methoxybenzyl)sulfamoyl)benzoate (example 5-10b). ¹H NMR(400 MHz, DMSO-d6): δ, ppm: 3.65 (s, 3H), 4.24 (s, 2H), 4.33 (s, 2H),6.71 (d, 2H, J=8 Hz), 6.95 (d, 2H, J=8 Hz), 7.14 (d, 2H, J=8 Hz). 7.73(d, 2H, J=8 Hz), 7.89 (d, 2H, J=8 Hz), 8.06 (d, 2H, J=8 Hz).

Example 5-39 4-(N-benzyl-N-(3,4-dimethoxybenzyl)sulfamoyl)benzoic acid

Prepared as in example 5-10 from (3,4-dimethoxyphenyl)methanamine,methyl 4-(chlorosulfonyl)benzoate (Example 5-10c) and benzyl chloride.MS (M−H, 440.10); ¹H NMR (400 MHz, CD₃OD): δ, ppm: 3.59 (s, 3H), 3.76(s, 3H), 4.30 (s, 2H), 4.36 (s, 2H), 6.51 (d, 1H, J=1.7 Hz), 6.60 (m,1H), 6.76 (d, 1H, J=8.2 Hz), 7.12 (m, 2H), 7.21 (m, 3H), 7.95 (d, 2H,J=8.6 Hz), 8.18 (d, 2H, J=8.6 Hz).

The compound had an IC₅₀ on hT2R14 bitter receptor of 0.678 μM.

Example 5-404-(N-(3,4-dimethoxybenzyl)-N-(4-methoxybenzyl)sulfamoyl)benzoic acid

Prepared as in example 5-10 from (3,4-dimethoxyphenyl)methanamine,methyl 4-(chlorosulfonyl)benzoate (Example 5-10c) and4-methoxybenzylbromide. MS (M−H, 470.10); ¹H NMR (400 MHz, DMSO-d6): δ,ppm: 3.49 (s, 3H), 3.67 (s, 3H), 3.68 (s, 3H), 4.20 (s, 2H), 4.22 (s,2H), 6.41 (d, 1H, J=1.4 Hz), 6.58 (dd, 1H, J1=8.2 Hz, J2=1.4 Hz), 6.78(m, 3H), 7.02 (d, 2H, J=8.6 Hz), 7.94 (d, 2H, J=8.4 Hz), 8.09 (d, 2H,J=8.4 Hz).

The compound had an IC₅₀ on hT2R14 bitter receptor of 1.47 μM.

Example 5-414-(N-(3-fluoro-4-methoxybenzyl)-N-(4-methoxybenzyl)sulfamoyl)-benzoicacid

Prepared as in example 5-10 from4-(bromomethyl)-2-fluoro-1-methoxybenzene and methyl4-(N-(4-methoxybenzyl)sulfamoyl)benzoate (example 5-10b). MS (M−H,458.10); ¹H NMR (400 MHz, CD₃OD): δ, ppm: 3.73 (s, 3H), 3.80 (s, 3H),4.24 (s, 2H), 4.28 (s, 2H), 6.75 (m, 4H), 6.88 (m, 1H), 6.97 (d, 2H,J=8.6 Hz), 7.88 (d, 2H, J=8.3 Hz), 8.14 (d, 2H, J=8.3 Hz).

The compound had an IC₅₀ on hT2R14 bitter receptor of 1.11 μM.

Example 5-424-((4-hydroxy-N-(2-methoxybenzyl)phenylsulfonamido)methyl)-benzoic acid

4-(N-(2-methoxybenzyl)sulfamoyl)phenyl acetate (example 5-42a) (50 mg,0.15 mmol) was dissolved in acetone (1.0 mL) followed by the addition ofcesium carbonate (97 mg, 0.30 mmol) and methyl 4-(bromomethyl)benzoate(38 mg, 0.17 mmol). The mixture was stirred at room temperatureovernight and then the inorganic salts were filtered off. Acetone wasremoved in vacuo and the residue was re-dissolved in ethyl acetate andwashed with water followed by brine. The organic layer was dried overmagnesium sulfate and concentrated. The crude product was purified bycolumn chromatography with ethyl acetate/hexanes as the eluent to affordmethyl4-((4-acetoxy-N-(2-methoxybenzyl)phenylsulfonamido)methyl)benzoate.

Methyl4-((4-acetoxy-N-(2-methoxybenzyl)phenylsulfonamido)methyl)benzoate(crude) was dissolved in THF (1.0 mL) and treated with aqueous NaOH (1N,2.0 mL, 2.0 mmol). The mixture was refluxed for an hour. Upon completionthe THF was removed in vacuo and the resulting aqueous solution wasacidified with 6 N aq HCl to a pH of ˜3. The aqueous phase was extractedwith EtOAc (2×15 mL) and the combined organic layers were washed withwater, brine, dried over Na₂SO₄ and concentrated. The crude product waspurified by reverse phase HPLC to afford 10.8 mg of the title compound(15% yield over two steps). MS (M−H, 426.1); ¹H NMR (400 MHz,acetone-d6): δ, ppm: 3.65 (s, 3H), 4.37 (s, 2H), 4.43 (s, 2H), 6.79 (m,2H), 7.01 (d, 2H, J=8.0 Hz), 7.17 (m, 2H), 7.28 (d, 2H, J=7.9 Hz), 7.73(d, 2H, J=8.0 Hz), 7.87 (d, 2H, J=7.9 Hz).

The compound had an IC₅₀ on hT2R14 bitter receptor of 2.56 μM.

Example 5-42a 4-(N-(2-methoxybenzyl)sulfamoyl)phenyl acetate

A solution of 4-(chlorosulfonyl)phenyl acetate (example 5-42b) (531 mg,2.265 mmol) in 5.0 mL of dichloromethane, was cooled to 0° C. in an icebath. (2-methoxyphenyl)methanamine (325 μL, 2.492 mmol) andtriethylamine (347 μL, 2.492 mmol) were added. The ice bath was thenremoved and the mixture warmed to ambient temperature and stirred for 2hours. The reaction mixture was concentrated and the crude product waspurified by column chromatography (hexanes/ethyl acetate=90/10 to 30/70)to afford pure 4-(N-(2-methoxybenzyl)sulfamoyl)phenyl acetate (743 mg,89%) as a white solid. MS (M+H, 336.1) ¹H NMR (400 MHz, CDCl₃): δ, ppm:2.32 (s, 3H), 3.71 (s, 3H), 4.18 (d, 2H, J=5.8 Hz), 5.15 (t, 1H, J=5.8Hz), 6.71 (d, 1H, J=8.2 Hz), 6.82 (br t, 1H, J=7.4 Hz), 7.07 (br d, 1H,J=7.4 Hz), 7.10 (d, 2H, J=8.7 Hz), 7.19 (br t, 1H, J=7.8 Hz), 7.74 (d,2H, J=8.7 Hz).

Example 5-42b 4-(chlorosulfonyl)phenyl acetate

6.285 g (36.08 mmol) of 4-hydroxybenzenesulfonic acid was dissolved in amixture of 30 mL of acetic anhydride and 15 mL of acetic acid andrefluxed for 6 hours. The volatiles were evaporated and placed underhigh vacuum overnight. The resulting crude product was, dissolved in 100mL of DCM and treated with 4.72 mL of oxalyl chloride (54.12 mmol) and139 μL of DMF (1.804 mmol) at 0° C. Stirring was continued until gasevolution ceased then the reaction was concentrated and re-dissolved inEtOAc. The organic layer was washed twice with 2 N H₂SO₄, and dried withbrine and MgSO₄. concentration gave 7.067 g of 4-(chlorosulfonyl)phenylacetate as a dark thick oil that eventually solidified (83% yield overtwo steps).

¹H-NMR (400 MHz, CDCl₃): δ, ppm: 2.35 (s, 3H), 7.37 (d, 2H, J=8.9 Hz),8.06 (d, 2 H, J=8.9 Hz). ¹³C-NMR (100 MHz; CDCl₃): δ, ppm: 21.13,123.05, 128.91, 141.15, 155.80, 168.29.

Example 5-434-((4-hydroxy-N-(3-methoxybenzyl)phenylsulfonamido)methyl)-benzoic acid

Prepared as in example 5-42 from (3-methoxyphenyl)methanamine and methyl4-(bromomethyl)benzoate. MS (M−H, 426.10); ¹H NMR (400 MHz, acetone-d6):δ, ppm: 3.66 (s, 3H), 4.32 (s, 2H), 4.40 (s, 2H), 6.65 (br s, 1H), 6.73(m, 2H), 7.05 (d, 2H, J=8.5 Hz), 7.12 (t, 1H, J=8.0 Hz), 7.27 (d, 2H,J=8.0 Hz), 7.82 (d, 2H, J=8.5 Hz), 7.88 (d, 2H, J=8.0 Hz).

The compound had an IC₅₀ on hT2R14 bitter receptor of 0.188 μM.

Example 5-444-((4-hydroxy-N-(4-methoxybenzyl)phenylsulfonamido)methyl)-benzoic acid

Prepared as in example 5-42 from (4-methoxyphenyl)methanamine and methyl4-(bromomethyl)benzoate. MS (M−H, 426.10); ¹H NMR (400 MHz, acetone-d6):δ, ppm: 3.73 (s, 3H), 4.27 (s, 2H), 4.35 (s, 2H), 6.76 (d, 2H, J=8.0Hz), 7.04 (d, 4H), 7.24 (d, 2H, J=7.8 Hz), 7.79 (d, 2H, J=8.2 Hz), 7.88(d, 2H, J=7.8 Hz).

The compound had an IC₅₀ on hT2R14 bitter receptor of 3.43 μM.

Example 5-454-((N-(3-fluorobenzyl)-4-hydroxyphenylsulfonamido)methyl)benzoic acid

Prepared as in example 5-42 from (3-fluorophenyl)methanamine and methyl4-(bromomethyl)benzoate. MS (M−H, 414.1), ¹H NMR (400 MHz, acetone-d6):δ, ppm: 4.37 (s, 2H), 4.42 (s, 2H), 6.94 (m, 3H), 7.06 (d, 2H, J=8.3Hz), 7.21 (m, 1H), 7.28 (d, 2H, J=7.6 Hz), 7.81 (d, 2H, J=8.3 Hz), 7.87(d, 2H, J=7.6 Hz).

The compound had an IC₅₀ on hT2R14 bitter receptor of 0.459 μM.

Example 5-46N-benzyl-N-(4-methoxybenzyl)-4-(1H-tetrazol-5-yl)benzene-sulfonamide

N-benzyl-4-cyano-N-(4-methoxybenzyl)benzenesulfonamide (example 5-46a,400 mg, 1 mmol) and trimethyltin azide (400 mg, 2 mmol) were dissolvedin toluene (10 mL) and microwaved at 150° C. for 3 hours. An additional2 equivalents of trimethyltin azide were added and the reaction wasmicrowaved at 150° C. for another 3 hr. The mixture was cooled andfiltered to give crude tin tetrazole which was hydrolyzed in MEOH/concHCl (50 mL:20 mL). Water was added and the resulting precipitate wascollected by filtration. The product was recrystallized with absoluteethanol and water to provideN-benzyl-N-(4-methoxybenzyl)-4-(1H-tetrazol-5-yl)benzene-sulfonamide asa white solid. MS (M+H, 436.10); ¹H NMR (400 MHz, DMSO-d6): δ, ppm: 3.65(s, 3H), 4.27 (s, 2H), 4.30 (s, 2H), 6.75 (d, J=8.4 Hz, 2H), 6.98 (d,J=8.4 Hz, 2H), 7.08 (m, 2H), 7.20 (m, 3H), 8.05 (d, J=8.4 Hz, 2H), 8.22(d, J=9.2 Hz, 2H).

The compound had an IC₅₀ on hT2R14 bitter receptor of 3.67 μM.

Example 5-46a N-benzyl-4-cyano-N-(4-methoxybenzyl)benzenesulfonamide

4-cyanobenzene-1-sulfonyl chloride (600 mg, 3 mmol) was added to asolution of N-benzyl-1-(4-methoxyphenyl)methanamine (example 5-46b, 750mg, 3.3 mmol) and triethylamine (500 mg, 3.6 mmol) in dichloromethane(15 mL). The reaction was stirred at ambient temperature for 4 hoursthen concentrated on the rotovap. The crude was purified on silica gelto afford N-benzyl-4-cyano-N-(4-methoxybenzyl)benzene-sulfonamide as awhite solid. ¹H NMR (400 MHz, DMSO-d6): δ, ppm: 3.68 (s, 3H), 4.26 (s,2H), 4.31 (s, 2H), 6.75 (d, J=8.4 Hz, 2H), 6.97 (d, J=9.2 Hz, 2H), 7.07(m, 2H), 7.21 (m, 3H), 7.98 (d, J=8.4 Hz, 2H), 8.04 (d, J=8.8 Hz, 2H).

Example 5-46b N-benzyl-1-(4-methoxyphenyl)methanamine

4-methoxybenzaldehyde (5 g, 35 mmol) and benzylamine (3.8 g, 35 mmol)were added to sodium triacetoxyborohydride (10.4 g, 49 mmol) indichloroethane (125 mL). The reaction was stirred at ambient temperature2 hours, then concentrated. The mixture was diluted with dichloromethane(200 mL), washed with saturated aqueous sodium hydrogen carbonate (200mL), brine (200 mL) and dried over magnesium sulfate. The crude aminewas concentrated and purified by silica gel chromatography (70% ethylacetate in hexanes) To afford N-benzyl-1-(4-methoxyphenyl)methanamine asan oil. ¹H NMR (400 MHz, DMSO-d6): δ, ppm: 3.59 (s, 2H), 3.64 (s, 2H),3.71 (s, 3H), 6.86 (d, J=8.8 Hz, 2H), 7.28 (m, 7H).

Example 5-47 2-(4-(N-benzyl-N-(4-methoxybenzyl)sulfamoyl)phenyl)aceticacid

Prepared as in example 5-27 from Methyl2-(4-(chlorosulfonyl)phenyl)acetate (example 5-47a),4-methoxybenzaldehyde and benzylamine. MS (M−H, 424.10); ¹H NMR (400MHz, DMSO-d6): δ, ppm: 3.66 (s, 3H), 3.72 (s, 2H), 4.18 (s, 2H), 4.22(s, 2H), 6.73 (d, J=8.4 Hz, 2H), 6.91 (d, J=8.4 Hz, 2H), 7.02 (m, 2H),7.19 (m, 3H), 7.49 (d, J=8.4 Hz, 2H), 7.80 (d, J=8 Hz, 2H).

Example 5-47a Methyl 2-(4-(chlorosulfonyl)phenyl)acetate

2-(4-(chlorosulfonyl)phenyl)acetic acid (600 mg, 2.6 mmol) was added tothionyl chloride (3 mL) and heated at 80° C. for 1 hour. The reactionmixture was concentrated, cooled to 0° C. in an ice bath and ice coldmethanol was added dropwise. The mixture was stirred for 30 minutes andconcentrated to afford Methyl 2-(4-(chlorosulfonyl)phenyl)-acetate as anoil. ¹H NMR (400 MHz, DMSO-d6): δ, ppm: 3.57 (s, 3H), 3.65 (s, 2H), 7.21(d, J=8 Hz, 2H), 7.55 (d, J=8 Hz, 2H).

Example 5-48 4-((N-benzyl-4-carboxyphenylsulfonamido)methyl)benzoic acid

Prepared as in example 5-10 from methyl-4-(chlorosulfonyl)benzoate(example 5-10c), benzyl amine and methyl 4-(bromomethyl)benzoate MS(M+H, 426.10); ¹H NMR (400 MHz, DMSO-d6): δ, ppm: 4.29 (s, 2H), 4.34 (s,2H), 7.03 (m, 2H), 7.12 (m, 2H), 7.68 (d, J=8 Hz, 2H), 7.94 (d, J=8.8Hz, 2H), 8.06 (d, J=8.8 Hz, 2H), 13.03 (s, 2H).

Example 5-49 4-(benzyl(4-methoxybenzyl)carbamoyl)benzoic acid

Prepared as in example 5-27 from 4-(chlorocarbonyl)benzoate andN-benzyl-1-(4-methoxyphenyl)methanamine. ¹H NMR (400 MHz, DMSO-d6): δ,ppm: 3.72 (d, J=7.6 Hz, 3H), 4.26 (s, 1H), 4.30 (s, 1H), 4.50 (s, 1H),4.55 (s, 1H), 6.89 (m, 2H), 7.02 (m, 1H), 7.10 (m, 1H), 7.10 (m, 1H),7.20 (m, 1H), 7.28 (m, 4H), 7.55 (m, 2H), 7.96 (m, 2H), 13.13 (s, 1H).

Example 5-504-(N-(4-fluoro-3-methoxybenzyl)-N-(4-methoxybenzyl)sulfamoyl)-benzoicacid

Prepared as in example 5-10 from4-(bromomethyl)-4-fluoro-3-methoxybenzene and methyl4-(N-(4-methoxybenzyl)sulfamoyl)benzoate (example 5-10b). MS (M−H,458.10); ¹H NMR (400 MHz, DMSO-d6): δ, ppm: 3.59 (s, 3H), 3.67 (s, 3H),4.25 (s, 2H), 4.25 (s, 2H), 6.62 (m, 2H), 6.77 (d, J=8.4 Hz, 2H), 7.02(m, 3H), 7.97 (d, J=8 Hz, 2H), 8.01 (d, J=8.8 Hz, 2H), 13.49 (s, 1H).

The compound had an IC₅₀ on hT2R14 bitter receptor of 1.65 μM.

Additional compounds were experimentally tested and found to have arelatively high level of effectiveness as inhibitors of hT2R14 bitterreceptor. The results of that testing are shown below in Table C.

TABLE C Compound hT2R14 No. Compound IC₅₀ (μM) 5-51

4-((N-(4-fluorobenzyl)thiophene-2-sulfonamido)methyl)cyclohexanecarboxylic acid 0.342 5-52

4-((N-(4-fluorobenzyl)pyridine-3-sulfonamido)methyl)cyclohexanecarboxylic acid 8.434 5-53

4-((N-(4-fluorobenzyl)pyridine-2-sulfonamido)methyl)cyclohexanecarboxylic acid 5-54

4-(N-(furan-2-ylmethyl)sulfamoyl)benzoic acid 5-55

4-(N-benzyl-N-methylsulfamoyl)benzoic acid 5-56

4-(N-(4-methoxybenzyl)sulfamoyl)benzoic acid 5-57

3-(N-benzyl-N-(4- methoxybenzyl)sulfamoyl)propanoic acid 5-58

4-(N-(2-methoxybenzyl)-N-(4- methoxybenzyl)sulfamoyl)benzoic acid 2.9435-59

3-(4-(N-benzyl-N-(4- methoxybenzyl)sulfamoyl)phenyl)propanoic acid 5-60

2-(N-benzyl-N-(4- methoxybenzyl)sulfamoyl)benzoic acid 5-61

4-(N-(4-methoxybenzyl)-N-(thiophen-2- ylmethyl)sulfamoyl)benzoic acid5-62

4-(N-(4-methoxybenzyl)-N-(thiophen-3- ylmethyl)sulfamoyl)benzoic acid5-63

4-(N-(4-methoxybenzyl)-N-((5- methylfuran-2-yl)methyl)sulfamoyl)benzoicacid 5-64

4-(N-(4-methoxybenzyl)-N-(2- methylbenzyl)sulfamoyl)benzoic acid 5-65

4-(N-(4-methoxybenzyl)-N-(3- methylbenzyl)sulfamoyl)benzoic acid 5-66

4-(N-(4-methoxybenzyl)-N-(4- methylbenzyl)sulfamoyl)benzoic acid 5-67

4-(N-(3,5-dimethoxybenzyl)-N-(4- methoxybenzyl)sulfamoyl)benzoic acid12.141 5-68

4-(N-(2-chlorobenzyl)-N-(4- methoxybenzyl)sulfamoyl)benzoic acid 7.0445-69

4-(N-(4-fluorobenzyl)-N-(4- methoxybenzyl)sulfamoyl)benzoic acid 8.2835-70

4-(N-benzyl-N-(4- methylbenzyl)sulfamoyl)benzoic acid 5.394 5-71

4-(N-benzyl-N-(2- methoxybenzyl)sulfamoyl)benzoic acid 5.061 5-72

4-(N-benzyl-N-(2- fluorobenzyl)sulfamoyl)benzoic acid 6.164 5-73

4-(N-benzyl-N-(3- methylbenzyl)sulfamoyl)benzoic acid 2.290 5-74

4-(N-benzyl-N-(3- methoxybenzyl)sulfamoyl)benzoic acid 5.991 5-75

4-(N-benzyl-N-(3- cyanobenzyl)sulfamoyl)benzoic acid 5-76

3-((N-benzyl-4- carboxyphenylsulfonamido)methyl)benzoic acid 5-77

4-(N-benzyl-N-(4- chlorobenzyl)sulfamoyl)benzoic acid 3.977 5-78

4-(N-benzyl-N-(4- (methylthio)benzyl)sulfamoyl)benzoic acid 2.458 5-79

4-(N-benzyl-N-(3- fluorobenzyl)sulfamoyl)benzoic acid 2.310 5-80

4-(N-benzyl-N-(2- methylbenzyl)sulfamoyl)benzoic acid 2.926 5-81

4-(N-benzyl-N-(4- methoxyphenethyl)sulfamoyl)benzoic acid 5-82

3-((4-carboxy-N-(4- methoxybenzyl)phenylsulfonamido)methyl) benzoic acid5-83

4-(N-benzyl-N-(2- chlorobenzyl)sulfamoyl)benzoic acid 4.486 5-84

4-(N-benzyl-N-(3- chlorobenzyl)sulfamoyl)benzoic acid 4.286 5-85

4-(N-benzyl-N-(4- ethylbenzyl)sulfamoyl)benzoic acid 8.580 5-86

4-(N-(4-methoxybenzyl)-N-(pyridin-4- ylmethyl)sulfamoyl)benzoic acid5-87

4-(N-(4-methoxybenzyl)-N-(pyridin-2- ylmethyl)sulfamoyl)benzoic acid5-88

4-(N-(4-methoxybenzyl)-N-(pyridin-3- ylmethyl)sulfamoyl)benzoic acid5-89

4-(N-benzyl-N-(4-methoxy-3- methylbenzyl)sulfamoyl)benzoic acid 1.9955-90

4-(N-benzyl-N-((6-methoxypyridin-3- yl)methyl)sulfamoyl)benzoic acid3.258 5-91

4-(N-benzyl-N-(4- cyanobenzyl)sulfamoyl)benzoic acid 1.848 5-92

4-(N-benzyl-N-(2- cyanobenzyl)sulfamoyl)benzoic acid 4.627 5-93

4-(N-(benzo[d][1,3]dioxol-5-ylmethyl)-N- benzylsulfamoyl)benzoic acid2.167 5-94

4-(3-benzyl-3-(4- methoxybenzyl)ureido)benzoic acid 5-95

4-(N-(4-methoxybenzyl)-N-(1- phenylethyl)sulfamoyl)benzoic acid 1.3975-96

4-(N-(4-methoxybenzyl)-N-(1,2,3,4- tetrahydronaphthalen-1-yl)sulfamoyl)benzoic acid 2.415 5-97

4-(N-((4- methoxyphenyl)(phenyl)methyl)sulfamoyl)benzoic acid 5-98

4-(4-benzyl-3-(4-methoxybenzyl)-2,5- dioxoimidazolidin-1-yl)benzoic acid5-99

4-(N-benzyl-N-(4- hydroxybenzyl)sulfamoyl)benzoic acid 5.207 5-100

4-((N-(2- methoxybenzyl)phenylsulfonamido)methyl) benzoic acid 1.2945-101

4-((N-(3- methoxybenzyl)phenylsulfonamido)methyl) benzoic acid 0.3455-102

4-((N-(4- methoxybenzyl)phenylsulfonamido)methyl) benzoic acid 2.2195-103

4-((N-(2- fluorobenzyl)phenylsulfonamido)methyl)- benzoic acid 0.4295-104

4-((N-(3- fluorobenzyl)phenylsulfonamido)methyl)- benzoic acid 0.4065-105

4-((N-(4- fluorobenzyl)phenylsulfonamido)methyl)- benzoic acid 0.9355-106

4-(2-methoxy-5H-dibenzo[c,e]azepin- 6(7H)-ylsulfonyl)benzoic acid 5-107

4-((N-(4-fluorobenzyl)-4- hydroxyphenylsulfonamido)methyl)benzoic acid4.086 5-108

4-(N-benzyl-N-(4- ethoxybenzyl)sulfamoyl)benzoic acid 5-109

4-(N-benzyl-N-(4- propoxybenzyl)sulfamoyl)benzoic acid 5-110

4-(N-benzyl-N-(4- isopropoxybenzyl)sulfamoyl)benzoic acid 5-111

4-((3-(3-chloro-4-methylphenyl)-1-(4-methoxybenzyl)thioureido)methyl)cyclohexanecarboxylic acid 6.136 5-112

4-((1-(4-methoxybenzyl)-3-(2-methoxyphenyl)thioureido)methyl)cyclohexanecarboxylic acid 8.852 5-113

4-((1-(4-ethylbenzyl)-3-o- tolylthioureido)methyl)cyclohexanecarboxylicacid 5.018 5-114

4-((3-(4-chloro-3-methylphenyl)-1-(2-methylbenzyl)thioureido)methyl)cyclohexanecarboxylic acid 4.872 5-115

ethyl 4-((4-acetamido-N-benzylphenylsulfonamido)methyl)cyclohexanecarboxylate 0.334 5-116

ethyl 4-((N-(3-bromobenzyl)-2,3- dihydrobenzo[b][1,4]dioxine-6-sulfonamido)methyl)cyclohexanecarboxylate 2.567 5-117

ethyl 4-((N-(2-chlorobenzyl)-4-methylphenylsulfonamido)methyl)cyclohexanecarboxylate 2.816 5-118

ethyl 4-((N-benzyl-4-bromophenylsulfonamido)methyl)cyclohexanecarboxylate 2.344 5-119

ethyl 4-((N-benzyl-4-chlorophenylsulfonamido)methyl)cyclohexanecarboxylate 0.672 5-120

4-((N-benzyl-4- chlorophenylsulfonamido)methyl)benzoic acid 0.394 5-121

4-(N-(2-methoxybenzyl)-N-(4- methoxybenzyl)sulfamoyl)benzoic acid 2.9435-122

N-benzyl-3-methoxy-N-(4- methoxybenzyl)benzenesulfonamide 2.219 5-123

4-((N-(4-fluorobenzyl)-4-methoxyphenylsulfonamido)methyl)cyclohexanecarboxylic acid 0.092 5-124

4-((4-acetamido-N-(4-fluorobenzyl)phenylsulfonamido)methyl)cyclohexanecarboxylic acid 0.8425-125

4-((4-amino-N-(4-fluorobenzyl)phenylsulfonamido)methyl)cyclohexanecarboxylic acid 1.6515-126

4-((4-methyl-N-(4-methylbenzyl)phenylsulfonamido)methyl)cyclohexanecarboxylic acid 0.1095-127

4-((N-(4-cyanobenzyl)-4-methylphenylsulfonamido)methyl)cyclohexanecarboxylic acid 0.499 5-128

4-((N-(3-methoxybenzyl)-4-methylphenylsulfonamido)methyl)cyclohexanecarboxylic acid 0.036 5-129

4-(N-(4-methoxybenzyl)-N-(2- methylbenzyl)sulfamoyl)benzoic acid 5.3165-130

4-(N-(2-chlorobenzyl)-N-(4- methoxybenzyl)sulfamoyl)benzoic acid 7.0445-131

4-(N-benzyl-N-(4- fluorobenzyl)sulfamoyl)benzoic acid 8.283 5-132

4-(N-benzyl-N-(4- methylbenzyl)sulfamoyl)benzoic acid 5.394 5-133

4-(N-benzyl-N-(2- methoxybenzyl)sulfamoyl)benzoic acid 5.061 5-134

4-(N-benzyl-N-(2- fluorobenzyl)sulfamoyl)benzoic acid 6.164 5-135

4-(N-benzyl-N-(3- methylbenzyl)sulfamoyl)benzoic acid 2.290 5-136

4-(N-benzyl-N-(3- methoxybenzyl)sulfamoyl)benzoic acid 5.991 5-137

4-(N-benzyl-N-(4- chlorobenzyl)sulfamoyl)benzoic acid 3.977 5-138

4-(N-benzyl-N-(4- (methylthio)benzyl)sulfamoyl)benzoic acid 2.458 5-139

4-(N-benzyl-N-(3- fluorobenzyl)sulfamoyl)benzoic acid 2.310 5-140

4-(N-benzyl-N-(2- methylbenzyl)sulfamoyl)benzoic acid 2.926 5-141

4-(N-benzyl-N-(2- chlorobenzyl)sulfamoyl)benzoic acid 4.486 5-142

4-(N-benzyl-N-(3- chlorobenzyl)sulfamoyl)benzoic acid 4.286 5-143

4-(N-benzyl-N-(4- ethylbenzyl)sulfamoyl)benzoic acid 8.580 5-144

4-(N-benzyl-N-(4-methoxy-3- methylbenzyl)sulfamoyl)benzoic acid 1.9955-145

4-(N-benzyl-N-((6-methoxypyridin-3- yl)methyl)sulfamoyl)benzoic acid3.258 5-146

4-(N-benzyl-N-(4- cyanobenzyl)sulfamoyl)benzoic acid 1.848 5-147

4-(N-benzyl-N-(2- cyanobenzyl)sulfamoyl)benzoic acid 4.627 5-148

4-(N-(benzo[d][1,3]dioxol-5-ylmethyl)-N- benzylsulfamoyl)benzoic acid2.167 5-149

4-(N-(4-methoxybenzyl)-N-(1- phenylethyl)sulfamoyl)benzoic acid 1.3975-150

4-(N-(4-methoxybenzyl)-N-(1,2,3,4-tetrahydronaphthalen-1-yl)sulfamoyl)benzoic acid 2.415 5-151

4-(N-benzyl-N-(4- hydroxybenzyl)sulfamoyl)benzoic acid 5.207 5-152

4-((N-(2- methoxybenzyl)phenylsulfonamido)methyl) benzoic acid 1.2945-153

4-((N-(3- methoxybenzyl)phenylsulfonamido)methyl) benzoic acid 0.3455-154

4-((N-(4- methoxybenzyl)phenylsulfonamido)methyl) benzoic acid 2.2195-155

4-((N-(2- fluorobenzyl)phenylsulfonamido)methyl)benzoic acid 0.429 5-156

4-((N-(3- fluorobenzyl)phenylsulfonamido)methyl)benzoic acid 0.406 5-157

4-((N-(4- fluorobenzyl)phenylsulfonamido)methyl)benzoic acid 0.935 5-158

4-(2-methoxy-5H-dibenzo[c,e]azepin- 6(7H)-ylsulfonyl)benzoic acid 3.5715-159

4-((N-(4-fluorobenzyl)-4- hydroxyphenylsulfonamido)methyl)benzoic acid4.086 5-160

4-(N-(2,4-difluorobenzyl)-N-(4- methoxybenzyl)sulfamoyl)benzoic acid1.255 5-161

4-(3-(4-methoxyphenyl)-3,4- dihydroisoquinolin-2(1H)- ylsulfonyl)benzoicacid 6.267 5-162

4-(6-methoxy-1-phenyl-3,4- dihydroisoquinolin-2(1H)- ylsulfonyl)benzoicacid 9.616 5-163

4-(7-methoxy-1-phenyl-3,4- dihydroisoquinolin-2(1H)- ylsulfonyl)benzoicacid 2.879

Example 6-1(Z)-3-(5-(2,5-dimethoxybenzylidene)-4-oxo-2-thioxothiazolidin-3-yl)propanoicacid

3-(4-oxo-2-thioxothiazolidin-3-yl)propanoic acid (200 mg, 1 mmol),2,5-dimethoxy benzaldehyde (168 mg, 1 mmol) and piperidine (0.3 mL) werecombined in ethanol (3 mL) and irradiated in the microwave at 100° C.for 10 minutes. The reaction was cooled, the solid was collected byfiltration, washed with ethyl acetate/hexanes (1/1) and recrystallizedfrom ethanol to afford 85% yield of the title compound (309 mg,brown-orange solid). MS (M+H, 284); ¹H NMR (400 MHz, DMSO-d6): δ, ppm:2.57 (t, 2H)), 3.74 (s, J=6.8 Hz, 3H), 3.84 (s, 3H), 4.19 (t, 2H), 6.90(s, 1H), 7.10 (s, 2H), 7.86 (s, 1H).

The compound had an IC₅₀ on hT2R14 bitter receptor of 2.75 μM.

Example 6-2(E)-2-cyano-3-(furan-2-yl)-N-(5-phenyl-1,3,4-thiadiazol-2-yl)acrylamide

5-phenyl-1,3,4-thiadiazol-2-amine (600 mg, 3.3 mmol), cyano acetic acid(300 mg, 3.6 mmol), and EDC-HCl (861 mg, 4.5 mmol) were stirred inacetonitrile (15 mL) at room temperature for 2 hours. The mixture wasdiluted with aqueous 1N HCl and the aqueous phase was extracted withethyl acetate. The combined organic extracts were dried over sodiumsulfate, filtered and concentrated in vacuo. The residue was trituratedwith ethyl acetate/hexanes (1/9) to afford2-cyano-N-(5-phenyl-1,3,4-thiadiazol-2-yl)acetamide as a white solidwhich was used without further purification.

312 mg of 2-cyano-N-(5-phenyl-1,3,4-thiadiazol-2-yl)acetamide and 150 uLof furan-2-carbaldehyde were mixed in 2.5 mL of DMF and heated in amicrowave at 150° C. for 15 min. 5 ml of H₂O was added and the resultingprecipitate was collected and washed with water (4×) to give the crudeproduct. Recrystallization from ethanol gave 180 mg of pure product as abrown solid. MS (M+H, 323); ¹H NMR (400 MHz, DMSO-d6): δ, ppm: 6.82 (m,1H)), 7.43 (d, 1H), 7.49 (m, 4H), 7.87 (m, 2H), 8.15 (s, 1H), 8.22 (br,s, 1H).

The compound had an IC₅₀ on hT2R14 bitter receptor of 0.499 μM.

Example 6-3N-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)-1,4-dimethyl-2,3-dioxo-1,2,3,4-tetrahydroquinoxaline-6-sulfonamide

1,4-dimethyl-2,3-dioxo-1,2,3,4-tetrahydroquinoxaline-6-sulfonyl chloride(example 6-3a, 450 mg, 1.6 mmol) was added to a stirred solution of the2,3-dihydrobenzo[b][1,4]dioxin-6-amine (215 mg, 1.4 mmol) andtriethylamine (170 mg, 1.7 mmol) in dichloromethane (10 mL). Thereaction was stirred at ambient temperature overnight then concentrated.The crude product was purified by silica gel chromatography (0-30% ethylacetate in hexanes) to provideN-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)-1,4-dimethyl-2,3-dioxo-1,2,3,4-tetrahydroquinoxaline-6-sulfonamideas a white solid. MS (M+H, 404.10); ¹H NMR (400MHz, DMSO-d6): δ, ppm:3.47 (s, 3H), 3.49 (s, 3H), 4.12 (m, 4H), 6.55 (m, 1H), 6.61 (d, J=2.4Hz, 1H), 6.69 (d, J=8.4 Hz, 1H), 7.53 (m, 1H), 7.59 (d, J=2 Hz, 1H),10.00 (s, 1H).

The compound had an IC₅₀ on hT2R14 bitter receptor of 7.71 μM.

Example 6-3a1,4-dimethyl-2,3-dioxo-1,2,3,4-tetrahydroquinoxaline-6-sulfonyl chloride

Chlorosulfonic acid (3 mL) was heated to 65° C. and1,4-dimethylquinoxaline-2,3(1H,4H)-dione (example 6-3b, 1 g, 5.5 mmol)was added in portions over 0.5 hour. The reaction mixture was stirredfor 4 hours then cooled to ambient temperature and poured slowly ontoice. The resulting precipitate was filtered and washed with water togive 1,4-dimethyl-2,3-dioxo-1,2,3,4-tetrahydroquinoxaline-6-sulfonylchloride as a white solid. ¹H NMR (400 MHz, DMSO-d6): δ, ppm: 3.50 (s,6H), 7.25 (m, 2H), 7.38 (m, 4H).

Example 6-3b 1,4-dimethylquinoxaline-2,3(1H,4H)-dione

To a solution of NaH (2.5 g) in DMF (200 mL) was addedquinoxaline-2,3(1H,4H)-dione (5 g) in portions, followed by the slowaddition of methyl iodide (3.8 mL). The reaction mixture was stirred atambient temperature for 4 hours, then water was added (200 mL) Theresulting precipitate was collected by filtration and washed with waterto afford 1,4-dimethylquinoxaline-2,3(1H,4H)-dione as a white solid in95% yield. ¹H NMR (400 MHz, DMSO-d6): δ, ppm: 3.50 (s, 6H), 7.25 (m,2H), 7.38 (m, 4H).

Example 6-41,4-dimethyl-2,3-dioxo-N-(4-(pyridin-2-ylmethyl)phenyl)-1,2,3,4-tetrahydroquinoxaline-6-sulfonamide

The compound is commercially available and was purchased through RyanScientific.

The compound had an IC₅₀ on hT2R14 bitter receptor of 6.14 μM.

Additional compounds were experimentally tested and found to have arelatively high level of effectiveness as inhibitors of hT2R14 bitterreceptor. The results of that testing are shown below in Table D.

TABLE D Compound hT2R14 No. Compound IC₅₀ (μM) 6-5

N-(benzo[d][1,3]dioxol-5-yl)-1,3-dimethyl-2,4-dioxo-1,2,3,4-tetrahydroquinazoline-6- sulfonamide 0.264 6-6

N-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)- 1,3-dimethyl-2,4-dioxo-1,2,3,4-tetrahydroquinoazoline-6-sulfonamide 0.706 6-7

N-(3-fluoro-2-methylphenyl)-1,3-dimethyl-2,4-dioxo-1,2,3,4-tetrahydroquinazoline-6- sulfonamide 0.935 6-8

N-(2,6-dimethylphenyl)-1,3-dimethyl-2,4-dioxo-1,2,3,4-tetrahydroquinazoline-6- sulfonamide 6-9

1,3-dimethyl-2,4-dioxo-N-o-tolyl-1,2,3,4-tetrahydroquinazoline-6-sulfonamide 1.726 6-10

N-(4-fluorophenyl)-1,4,7-trimethyl-2,3-dioxo-1,2,3,4-tetrahydroquinoxaline-6- sulfonamide 12.623 6-11

N-ethyl-N-m-tolyl-2-(N,1,4-trimethyl-2,3-dioxo-1,2,3,4-tetrahydroquinoxaline-6- sulfonamido)acetamide 14.926 6-12

(Z)-3-(4-oxo-2-thioxo-5-(2,4,6- trimethoxybenzylidene)thiazolidin-3-yl)propanoic acid 9.371 6-13

(Z)-3-(5-(3,5-dimethoxybenzylidene)-4-oxo-2-thioxothiazolidin-3-yl)propanoic acid 5.378 6-14

(E)-3-(benzo[d][1,3]dioxol-5-yl)-2-cyano-N-(5-phenyl-1,3,4-thiadiazol-2- yl)acrylamide 2.906 6-15

7-cyclopentyl-1,3-dimethyl-5-(2-oxopropylthio)pyrimido[4,5-d]pyrimidine- 2,4(1H,3H)-dione 5.428 6-16

N-cyclohexyl-1,3-dimethyl-2,4-dioxo- 1,2,3,4-tetrahydroquinazoline-6-sulfonamide 7.239 6-17

N-(2-cyano-3,5-dimethylphenyl)-1,3- dimethyl-2,4-dioxo-1,2,3,4-tetrahydroquinazoline-6-sulfonamide 1.946 6-18

N-(3-fluoro-2-methylphenyl)-1,3-dimethyl-2,4-dioxo-1,2,3,4-tetrahydroquinazoline-6- sulfonamide 0.935 6-19

N-(2,3-dimethylquinoxalin-6-yl)-1,4- dimethyl-2,3-dioxo-1,2,3,4-tetrahydroquinoxaline-6-sulfonamide 6.332 6-20

1,3-dimethyl-2,4-dioxo-N-o-tolyl-1,2,3,4-tetrahydroquinazoline-6-sulfonamide 1.726 6-22

N-ethyl-N-methyl-2-(2,6,8-trimethyl-5,7-dioxo-5,6,7,8-tetrahydropyrimido[4,5- d]pyrimidin-4-ylthio)acetamide0.861 6-23

N-(4-chloro-2-fluorophenyl)-1,3-dimethyl-2,4-dioxo-1,2,3,4-tetrahydroquinazoline-6- sulfonamide 9.703 6-24

1,3,7-trimethyl-5-(2-oxo-2-(piperidin-1-yl)ethylthio)pyrimido[4,5-d]pyrimidine- 2,4(1H,3H)-dione 0.292 6-25

1,3,7-trimethyl-5-(2-morpholino-2-oxoethylthio)pyrimido[4,5-d]pyrimidine- 2,4(1H,3H)-dione 1.006 6-26

7-ethyl-1,3-dimethyl-5-(2-(4- methylpiperidin-1-yl)-2-oxoethylthio)pyrimido[4,5-d]pyrimidine- 2,4(1H,3H)-dione 0.721 6-27

ethyl 4-(2-(2-ethyl-6,8-dimethyl-5,7-dioxo-5,6,7,8-tetrahydropyrimido[4,5-d]pyrimidin-4-ylthio)acetyl)piperazine-1-carboxylate 0.763 6-28

5-(2-(azepan-1-yl)-2-oxoethylthio)-1,3- dimethylpyrido[2,3-d]pyrimidine-2,4(1H,3H)-dione 2.377 6-29

N-benzyl-2-(1,3-dimethyl-2,4-dioxo-1,2,3,4-tetrahydropyrido[2,3-d]pyrimidin-5- ylthio)acetamide 0.710 6-30

N-methyl-2,3-dioxo-1,2,3,4- tetrahydroquinoxaline-6-sulfonamide 6.234

TABLE 7 Representative screening results against 23 bitter receptors.Compounds from examples 5 and 6 above were screened against a panel of23 bitter receptors. The bitter receptors were activated to EC80 withthe corresponding agonist then treated with the above compounds at aconcentration of 25 μM. The data is summarized in the table below. 80%or greater inhibition = ++++ 60% to 80% inhibition = +++ 40% to 60%inhibition = ++ 20% to 40% inhibition = + Example 1 3 4 5 7 8 9 10 13 1416 44 5-51 ++ + + + ++++ + ++ 5-25 + +++ 5-53 + + ++ 5-22 ++ ++ + +++++ + ++ 5-11 +++ +++ + ++ + ++ ++++ ++ +++ 5-24 + + + 5-37 +++ + +++++ + + 5-30 + ++ 275 + + + ++ + ++++ + +++ 5-60 5-46 + + ++++ + ++5-59 5-47 + + 5-28 + ++++ + + 5-1 +++ ++ ++ + +++ + ++++ +++ +++ 5-54 +5-5 ++ + ++ + + ++++ ++ ++ 5-56 + 5-49 + + 5-20 ++++ + + + ++ + ++++ +++++ 5-21 + + ++++ + ++ 5-3 ++ +++ + ++ ++++ + ++ 5-8 + ++ ++ ++++ + ++5-4 + ++ ++ 5-29 5-2 ++ + + ++ +++ ++++ +++ +++ 5-7 + + + + ++ ++5-6 + + + ++ ++++ + ++ 5-35 + + + + + + 5-81 + + + ++++ + ++ 5-55 + +++++ 5-61 + 5-63 + + 5-62 + + + + 5-76 + + + 5-77 ++ + + + + ++++ + +5-78 + + + ++++ + + 5-80 ++ + + + + ++++ + + 5-79 ++ + + ++++ + ++5-69 + + + + ++++ + 5-70 ++ ++ + + ++ +++ ++ + 5-48 5-75 + 5-72 + ++ + +++++ + + 5-71 + + + + ++++ + 5-73 ++ + + ++ ++++ + 5-74 + + + ++++++ + + 5-1 +++ ++ ++ +++ + ++++ ++ ++ 5-1 +++ ++ ++ + + +++ + ++++ ++++ 5-65 + 5-68 + ++ + + ++++ + ++ 5-64 + + + + + 5-34 ++ 734 ++ + + ++++++ + ++ 5-67 ++ + + ++ +++ + ++ 5-31 + + + 5-66 + + + + 5-14 + +++++++ +++ + ++ ++++ + ++++ +++ +++ 5-23 +++ + ++ + ++++ + ++++ ++ +++ 5-18+++ ++ +++ 5-19 + ++++ ++ 5-25 +++ ++ ++ + +++ ++++ ++ +++ 5-38 + 5-825-10 +++ 477 ++ ++ ++++ ++ ++++ ++++ ++ 5-12 +++ 371 ++ ++ ++ ++++ +++++ +++ +++ 5-1 +++ 201 ++ ++ + +++ + ++++ +++ + 5-83 ++ + + ++ ++++++ + 5-84 ++ + + ++++ + + 5-85 ++ + + ++ + ++++ ++ 5-87 5-88 + 345 + +5-89 ++ + ++ + +++ + ++++ + +++ 5-90 ++ + +++ ++ ++++ + +++ 5-86 + +5-58 +++ + +++ +++ ++++ + +++ 5-17 + + +++ ++++ + +++ 5-37 + ++++ +++5-1 ++++ + ++ + ++++ + ++++ ++ +++ 5-16 ++ + + + +++ 5-92 + + + + ++++ +++++ 5-91 +++ + ++ ++++ + +++ 5-1 +++ + ++ ++ ++ +++ + ++++ ++ +++ 5-13++ + + + + ++++ + +++ 5-15 + + + + ++ +++ + +++ 5-19 + + + ++++ + ++5-16 +++ + + + + + + +++ 5-1 +++ ++ + ++ ++ +++ + ++++ ++ +++ 5-17 + + ++++ 5-1 ++ ++ ++ ++ + ++++ +++ +++ 5-30 ++ 396 + + ++ + ++++ ++ +++5-96 + + + ++++ + +++ 5-95 ++ + + + ++++ ++ +++ 5-97 + +++ + 5-50 ++565 + + + + ++++ ++ +++ 5-98 + ++ +++ 5-1 +++ + ++ + +++ + ++++ ++ +++5-48 ++ 410 + +++ ++++ + +++ 5-57 + + 5-1 +++ + + + +++ + ++++ ++ +++5-94 + ++ 5-107 +++ + 5-45 ++ ++++ + ++ 5-44 + ++++ ++ 5-43 + + ++++ + +5-42 + ++++ + ++ 5-40 + + + ++ ++++ + +++ 5-41 ++ 332 + ++ + ++++ + +++5-1 + ++ ++ ++ + +++ + ++++ ++ ++++ 5-33 ++ ++ ++ + + +++ + ++++ ++ +++5-23 +++ ++ ++ ++ ++ ++++ + ++++ +++ ++++ 5-27 ++ ++ + +++ + ++++ + +++5-26 ++ + + 5-93 + ++ + + ++ ++++ ++ +++ 5-99 310 + ++++ + +++5-105 + + + + + ++++ ++ ++ 5-104 ++ ++ ++ ++ + ++ ++++ + +++ 5-1 ++ ++++ + +++ ++++ ++ +++ 5-103 ++ ++ + + + ++++ + +++ 5-102 ++ ++ + ++ +++++ + +++ 5-101 +++ ++ ++ +++ + +++ ++++ ++ +++ 5-100 + ++ + + + + ++ ++++ 5-1 ++ + + ++ + +++ ++++ ++ +++ 5-93 +++ ++++ + ++ +++ + ++++ ++ +++5-36 ++ + + ++++ + ++ 5-106 ++++ + ++ 5-108 ++ +++ + + ++ ++++ + +++5-109 ++ ++ + ++ + ++++ + ++ 5-110 ++ +++ + ++ + + ++ + ++++ + +++ 6-2331 ++++ 6-9 ++ ++++ + 6-3 + ++++ + 6-8 + 6-7 + ++ 6-11 + + ++ +6-10 + + + ++ ++ + 6-4 + + ++++ ++++ + 6-5 717 ++++ ++ 6-1 + 3866  932++++ ++++ + ++ 6-6 895 ++++ Example 51 54 55 61 63 64 65 67 71 75 5-51+++ ++++ +++ ++ + ++ + 5-25 ++ + ++ + 5-53 ++ ++ + 5-22 +++ ++++ +++ ++++ + 5-11 ++++ ++++ +++ ++ + + +++ ++ 5-24 ++ ++++ + +++ + 5-37 ++++ + ++++ + 5-30 + ++++ ++ ++++ ++++ + + +++ + 5-60 + 5-46 + ++++ +++ ++ +++++ 5-59 ++ + + 5-47 +++ + + 5-28 ++ + ++++ ++ ++++ ++++ + ++ + 5-1+++ + +++ ++++ ++++ +++ +++ ++ +++ + 5-54 + 5-5 + + ++++ ++++ +++++++ + + +++ + 5-56 + 5-49 ++++ +++ + ++ + 5-20 ++++ ++++ ++ +++ + ++ ++5-21 + +++ +++ + + 5-3 + ++ ++++ ++ +++ ++++ + + 5-8 ++ + ++++ +++ ++++++ ++ ++ + 5-4 ++++ + +++ ++++ + 5-29 +++ ++ 5-2 ++ + ++++ ++++ +++++++ + + +++ + 5-7 ++++ ++++ ++++ ++++ + ++ + 5-6 ++ + ++++ ++++ ++++++++ + ++ + 5-35 + +++ ++++ +++ + ++ 5-81 + +++ +++ +++ ++ + + + 5-55++ + ++++ ++ 5-61 + ++ ++++ + + 5-63 +++ +++ ++ + + 5-62 +++ ++ ++++++ + + 5-76 ++ ++ + + 5-77 ++ ++ ++ +++ +++ ++ ++ + ++ 5-78 + + +++ +++++++ ++ +++ + ++ + 5-80 ++ + +++ +++ ++++ +++ ++ + +++ + 5-79 ++++ + +++++++ ++++ +++ ++ + +++ 5-69 ++ +++ +++ ++++ ++ + + 5-70 ++ ++ +++ ++++++ + ++ + ++ + 5-48 ++++ +++ 5-75 + + + + 5-72 + + ++ +++ ++++ + + + +++ 5-71 ++ +++ ++ ++++ ++++ + ++ + ++ + 5-73 ++++ +++ ++ +++ ++++ + ++ +++ + 5-74 ++ ++ +++ ++++ ++++ + ++ + +++ 5-1 +++ ++ +++ ++++ ++++ ++++ + +++ + 5-1 ++++ + ++++ ++++ ++++ +++ +++ ++ +++ + 5-65 5-68 + +++++ +++ ++++ +++ + + +++ + 5-64 + + + 5-34 + +++ ++++ ++++ ++++ +++++ + +++ 5-67 ++ ++++ ++ ++++ +++ + + ++ 5-31 5-66 5-14 +++ ++++ ++++++++ ++++ +++ ++++ ++ ++++ ++ 5-23 +++ ++ ++++ ++++ ++++ ++++ ++++ +++++ ++ 5-18 ++ ++++ +++ +++ ++++ + ++ 5-19 + ++++ + +++ ++++ + + 5-25 ++++ +++ ++++ ++++ ++++ ++++ ++ ++++ 5-38 ++++ ++++ + + 5-82 ++++++++ + + 5-10 ++++ ++++ +++ ++++ ++++ +++ ++++ +++ +++ + 5-12 ++++ ++++++++ ++++ ++++ +++ ++++ +++ ++++ + 5-1 ++++ +++ +++ ++++ ++++ + ++++++ + + 5-83 + ++ +++ ++++ +++ + +++ ++ + ++ 5-84 +++ ++ +++ ++++ +++ ++++ + + + 5-85 + +++ +++ ++++ +++ +++ ++ + + 5-87 ++ + + + 5-88 +++++ + + + 5-89 ++++ + ++++ ++++ ++++ +++ ++++ + +++ + 5-90 +++ ++++ +++++++ ++ +++ 5-86 +++ +++ 5-58 + +++ +++ ++++ ++++ +++ ++++ + ++++ + 5-17++ ++++ ++++ ++++ ++++ ++ ++ + 5-37 ++++ +++ + +++ ++ +++ 5-1 +++ ++++++ ++++ ++++ +++ ++ + ++++ ++ 5-16 ++++ +++ ++ ++++ ++ + 5-92 ++++++++ ++++ ++++ ++ 5-91 + ++++ ++++ ++++ ++++ + +++ 5-1 +++ ++ ++++ ++++++++ ++ +++ + +++ + 5-13 + +++ ++++ ++++ ++++ +++ + ++ 5-15 ++++ +++++++ +++ ++ 5-19 + ++++ +++ ++ +++ ++ 5-16 ++++ ++++ +++ +++ + ++ 5-1 +++++ ++++ ++++ ++++ +++ +++ ++ +++ ++ 5-17 ++ ++++ ++++ ++ ++ + + 5-1 ++++++ ++++ ++++ ++++ +++ +++ ++ +++ + 5-30 ++ +++ ++++ ++++ ++++ ++++ +++++ +++ 5-96 ++++ ++++ ++++ +++ ++++ + ++ 5-95 + ++++ ++++ ++++ +++ +++ +++ + 5-97 + +++ + 5-50 + ++++ ++++ ++++ +++ ++ ++ +++ 5-98 ++++ ++++ +++5-1 +++ ++ ++++ ++++ ++++ +++ +++ ++ +++ + 5-48 ++ ++++ ++++ ++++ +++++ + +++ 5-57 +++ ++++ ++ 5-1 +++ ++ ++++ ++++ ++++ +++ +++ + ++++ +5-94 + +++ + 5-107 + ++++ +++ +++ + 5-45 ++ ++++ ++++ +++ + +++ 5-44 +++++ ++++ ++ ++ 5-43 + ++++ ++++ +++ ++ 5-42 ++++ ++ ++ ++ + 5-40 + +++++ ++++ ++++ +++ ++ 5-41 + +++ ++++ ++++ +++ ++ 5-1 +++ ++ ++++ +++++++ +++ +++ ++ +++ + 5-33 ++ +++ ++++ ++++ +++ +++ +++ ++ +++ 5-23 +++++ ++++ ++++ +++ +++ ++++ ++ ++++ 5-27 ++ ++ ++++ ++++ +++ +++ ++ +++++ + 5-26 + +++ +++ + 5-93 ++ +++ ++++ ++++ ++++ ++++ ++++ ++ +++5-99 + ++++ ++++ +++ ++ ++ +++ 5-105 + ++++ ++++ ++ ++++ +++ ++ +++ +5-104 + + +++ ++++ ++ ++++ +++ ++ +++ ++ 5-1 +++ ++ ++++ ++++ ++++ +++++++ ++ +++ 5-103 + ++ ++++ ++++ ++ ++++ ++ + ++++ + 5-102 + + ++++++++ + +++ ++ + +++ + 5-101 ++ ++ +++ ++++ +++ ++++ +++ ++ +++ ++5-100 + ++++ +++ + ++++ ++ +++ + 5-1 +++ ++ +++ ++++ ++++ ++++ +++ +++++ + 5-93 ++ +++ ++++ ++++ ++++ ++++ +++ ++ +++ 5-36 + ++++ ++++ +++++++ ++ +++ 5-106 ++ ++++ ++ ++++ +++ + + 5-108 ++ + ++++ ++++ +++ ++++++ + +++ 5-109 ++ + ++++ +++ ++ ++++ +++ + +++ 5-110 ++ + ++++ ++++ ++++++ +++ + +++ 6-2 ++ + + 6-9 + + 6-3 + + 6-8 6-7 + 6-11 + + 6-10 + +6-4 + + 6-5 + ++++ 278 ++ +++ 6-1 + ++ + +++ + + + 6-6 + 210 447 *

Example 7 Sensory Data for Example 10-10

To determine the effectiveness of an individual antagonist, taste testswere performed with a T2R8 specific agonist, the compound of interestand a reference bitter blocker. We have previously described a goodhT2R8 antagonist that was proven to have taste effect, Example 4-8 fromU.S. Provisional Appl. Ser. No. 60/957,129, filed Aug. 21, 2007:N-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)benzo[d][1,3]dioxole-5-carboxamide.It was shown to reduce bitterness of coffee by itself and in combinationwith a Broad spectrum bitter blocker). As shown in Table 6, whencompared to our control antagonist (Example 10-8) the hT2R8 antagonistof example 10-10 shows a greater ability to block perceived bitterness.

TABLE 6 Taste test results comparing a control bitter blocker and a morepotent bitter blocker 10-10. HTS assay Selected as Antagonist ExampleIC₅₀ more bitter conc. Number μM 10-8 +other p value □M 10-10 0.02-0.0414 2 0.004 1

As demonstrated by the taste tests of this example, the perception ofbitterness may be reduced or eliminated by incorporation of antagonistsof hT2R8 and the antagonist of Example 10-10 appears to be a more potentanalog when compared to known bitter antagonists likeN-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)benzo[d][1,3]dioxole-5-carboxamide(7767). It follows that the perception of bitterness may be reduced oreliminated by incorporation of antagonists of hT2R8 in compositions suchas foods, beverages and/or medicaments where bitter taste is elicited byT2R8 agonists.

Example 8 Identification of Antagonists of hT2R8

To identify antagonists, cell lines stably expressing hT2R8 togetherwith the promiscuous chimeric G16g44 protein were generated as describedin previous patent applications. A high-throughput assay was establishedusing the stable cell lines and FLIPR (Fluorescent Imaging PlateReader). An agonist of hT2R8 was used to activate the receptors up to70-80% of their respective maximal activity. For hT2R8, the agonist usedwas andrographolide (200 μM). To identify antagonists, compounds withdiverse chemical structures were added together with the agonist.Compounds that cause statistically significant reduction of the receptoractivity are pooled together, and reconfirmed with dose-dependentinhibition curves. scaffold A and scaffold B were identified as hT2R8antagonists (FIG. 1). Specific examples are presented in Table 1.

Example 9 hT2R8 Antagonists Example 9-12-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)isoindoline-1,3-dione

2-(1H-pyrazol-4-yl)isoindoline-1,3-dione (example 9-1a) (1.5 g, 7 mmol),4-(chloromethyl)-3,5-dimethylisoxazole (1.5 g, 10 mmol), and cesiumcarbonate (3.3 g, 10 mmol) were stirred in DMF (20 mL) at 80° C. for 3hours. The reaction was cooled, diluted with H₂O (150 mL), and extractedwith ethyl acetate (3×, 75 mL). The combined organic extracts were driedover sodium sulfate, filtered and concentrated on the rotovap. The solidproduct was triturated with ethyl acetate/hexanes (1/9) andre-crystallized from refluxing absolute ethanol (30 mL) to afford2-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)isoindoline-1,3-dione(900 mg, 38%) as a bright, light yellow solid. ¹H NMR (DMSO-d₆, 400 MHz)1.36 (d, 3H, δ J2.16 (s, 3H), 2.41 (s, 3H),=7.2 Hz), 5.22 (s, 2H), 7.81(s, 1H), 7.91-7.83 (m, 4H), 8.21 (s, 1H). MS M+H calculated 323.11;found 323.1. Melting point: 170-171° C. The title compound was shown toinhibit hT2R08 bitter receptor and had an IC₅₀ of 0.18 μM.

Example 9-1a 1-tosyl-1H-pyrazol-4-amine

1-tosyl-1H-pyrazol-4-amine (example 9-1b) (3 g, 12.7 mmol) andisobenzofuran-1,3-dione (1.9 g, 13 mmol) were stirred inDMF/Acetonitrile (1/1) (20 mL) at 100° C. for 1 hour. The mixture wascooled and diluted with H₂O. The precipitate was collected byfiltration, washed with additional water followed by ethyl acetate andhexanes. The solid product was dried under high vacuum to afford2-(1H-pyrazol-4-yl)isoindoline-1,3-dione (2.5 g, 92%) as a yellow solid.MS M+H calculated 214.1; found 214.1. ¹H NMR (CDCl₃, 400 MHz) δ7.93-8.10 (m, 6H), 13.03 (bs, 1H).

Example 9-1b 1-tosyl-1H-pyrazol-4-amine

4-nitro-1-tosyl-1H-pyrazole (example 9-1c) (3 g, 11.2 mmol) and 10%palladium on carbon (800 mg) in MeOH (150 mL) were stirred under 2atmospheres of hydrogen on the Parr hydrogenator for 3 hours. Themixture was filtered through celite, concentrated and purified by silicagel chromotography (80% ethyl acetate in hexanes) to afford1-tosyl-1H-pyrazol-4-amine (1.9 g, 71%) as a pink solid. ¹H NMR (CDCl₃,400 MHz),

2.40 (s, 3H), 3.01 (bs, 2H), 7.29 (d, 2H, J=8 Hz), 7.41 (d, 1H, J=1.2Hz), 7.53 (d, 1H, J=1.2 Hz), 7.81 (d, 2H, J=8 Hz).

Example 9-1c 4-nitro-1-tosyl-1H-pyrazole

4-nitro-1H-pyrazole (500 mg, 4.4 mmol), 4-methylbenzene-1-sulfonylchloride (840 mg, 4.4 mmol), and triethylamine (510 mg, 5 mmol) werestirred in DMF (25 mL) at 80° C. for 1 hour. The reaction was cooled,diluted with H₂O (200 mL) and extracted with ethyl acetate (3×, 100 ml).The organic phase was washed with aqueous 1N HCl (200 mL) and H₂O (200mL), dried over sodium sulfate, filtered and concentrated on therotovap. The solid was triturated with hexanes to afford4-nitro-1-tosyl-1H-pyrazole (600 mg, 50%) as an off white solid. ¹H NMR(DMSO-d₆, 400 MHz)

2.40 (s, 3H), 7.52 (d, 2H, J=8.4 Hz), 7.98 (d, 2H, J=8.8 Hz), 8.57 (s,1H), 9.58 (s, 1H).

Example 9-22-((1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-ylamino)methyl)benzonitrile

1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-amine-hydrochloride(100 mg, 0.44 mmol), 2-(bromomethyl)benzonitrile (115 mg, 0.6 mmol), andtriethylamine (0.5 mL, 3.5 mmol) in DMF (3 mL) were irradiated andstirred in the microwave reactor at 80° C. for 10 minutes. The reactionwas cooled, diluted with H₂O (50 mL) and extracted with ethyl acetate(3×, 30 mL). The combined organic extracts were dried over sodiumsulfate and concentrated on the rotovap. The residue was dissolved inethanol (70 mL), H₂O (3 mL) and acetic acid (1 mL) and the mixture wasrefluxed for 2 hours. The solution was concentrated on the rotovap,taken up in methanol (3 mL) and purified by reversed phase HPLC in 3-1mL aliquots (5 to 95% acetonitrile in H₂O: 16 minute gradient). The purefractions were combined and concentrated on the rotovap. The residue wastriturated with ethyl acetate/hexanes (1/9) to afford2-(1-(3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)isoindolin-1-one(85 mg, 61%) as a pure white solid. ¹H NMR (DMSO-d₆, 400 MHz): δ2.18 (s,3H), 2.43 (s, 3H), 4.97 (s, 2H), 5.18 (s, 2H), 7.58-7.68 (m, 3H), 7.77(s, 1H), 8.10 (d, J=8 Hz, 1H), 8.27 (s, 1H), 9.19 (bs, 1H). The titlecompound was shown to inhibit hT2R08 bitter receptor and had an IC₅₀value greater than 30 μM.

Example 9-32-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)isoindolin-1-one

2-((1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-ylamino)methyl)benzonitrile(example 9-2) (30 mg, 0.14 mmol) was stirred in a mixture of MeOH/2Naqueous NaOH (5 mL) at 100° C. for 30 minutes in the microwave reactor.The reaction was acidified with 1N aqueous HCl (100 mL) and extractedwith ethyl acetate (3×, 70 mL). The combined organic extracts were driedover sodium sulfate and concentrated. The residue was dissolved in MeOH(3 mL) and purified by reversed phase HPLC in 2-1.5 mL aliquots (5 to95% acetonitrile in H₂O: 16 minute gradient). The pure fractions werecombined and concentrated to afford2-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)isoindolin-1-one(21 mg, 50%) as a pure white solid. ¹H NMR (DMSO-d₆, 400 MHz): δ 2.15(s, 3H), 2.41 (s, 3H), 4.81 (s, 2H), 5.17 (s, 2H), 7.48-7.50 (m, 1H),7.51-7.52 (m, 2H), 7.72 (d, J=7.2 Hz, 1H), 7.75 (s, 1H), 8.20 (s, 1H).The title compound was shown to inhibit hT2R08 bitter receptor and hadan IC₅₀ of 11 μM.

Example 9-43-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)quinazoline-2,4(1H,3H)-dione

1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-amine hydrochloride(75 mg, 0.33 mmol), methyl 2-isocyanatobenzoate, and triethylamine (200mg, 2 mmol) in acetonitrile (3 mL) were irradiated in the microwavereactor at 100° C. for 30 minutes. The reaction was cooled, diluted withH₂O (75 mL), and extracted with ethyl acetate (3×, 50 mL). The combinedorganic extracts were dried over sodium sulfate, filtered andconcentrated on the rotovap. The residue was purified by silica gelchromotography (75% ethyl acetate in hexanes) to afford3-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)quinazoline-2,4(1H,3H)-dione(20 mg, 18%) as a light pink solid. ¹H NMR (DMSO-d₆, 400 MHz): δ2.16 (s,3H), 2.40 (s, 3H), 5.17 (s, 2H), 7.17-7.22 (m, 2H), 7.50 (s, 1H), 7.66(dt, J=8, 1.2 Hz, 1H), 7.92 (d, J=6.8 Hz, 1H), 7.95 (s, 1H), 11.52 (bs,1H). The title compound was shown to inhibit hT2R08 bitter receptor andhad an IC₅₀ of 1.5 μM.

Example 9-51-benzyl-3-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)tetrahydropyrimidin-2(1H)-one

1-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)tetrahydropyrimidin-2(1H)-one(example 9-5a) (50 mg, 0.18 mmol) and 60% sodium hydride (8 mg, 0.20mmol) in DMF (3 mL) were stirred at room temperature for 15 minutes thencooled to 0° C. Benzyl bromide (31 mg, 0.18 mmol) was added to themixture and allowed to warm up at room temperature then stirred for 2hours. The reaction was quenched with methanol and concentrated. Thereaction was diluted with brine (50 mL) and extracted withdichloromethane (2×, 50 mL). The combined organic extracts were driedover magnesium sulfate, filtered and concentrated on the rotovap. Theresidue was taken up in dichloromethane (5 mL) and purified by silicacolumn chromotography (100% to 90% dichloromethane in methanol: 30minute gradient) to afford1-benzyl-3-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)tetrahydropyrimidin-2(1H)-one(20 mg, 30%) as a white solid. ¹H NMR (DMSO-d₆, 400 MHz): δ1.85-1.91 (m,2H), 2.10 (s, 3H), 2.37 (s, 3H), 3.12 (m, 2H), 3.55 (t, J=5.8 Hz, 2H),4.48 (s, 2H), 5.05 (s, 2H), 7.22-7.31 (m, 5H), 7.49 (s, 1H), 7.84 (s,1H). LC/MS; [M+H] calculated for C₂₀H₂₃N₅O₂; expected 366.19; found366.15. The title compound was shown to inhibit hT2R08 bitter receptorand had an IC₅₀ of 1.65 μM.

Example 9-5a1-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)tetrahydropyrimidin-2(1H)-one

1-(3-chloropropyl)-3-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)urea(example 9-5b) (200 mg, 0.64 mmol) and 60% sodium hydride (28 mg, 0.71mmol) in DMF (2 mL) were stirred at 0° C. for 15 minutes then allowed towarm up to room temperature and stir for 2 hours. The reaction wasquenched with methanol and concentrated. The reaction was diluted withbrine (50 mL) and extracted with dichloromethane (2×, 50 mL). Thecombined organic extracts were dried over magnesium sulfate, filteredand concentrated. The residue was taken up in dichloromethane (5 mL) andpurified by silica column chromotography (100% to 90% dichloromethane inmethanol: 30 minute gradient) to afford1-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)tetrahydropyrimidin-2(1H)-one(84 mg, 48%) as a white solid. ¹H NMR (DMSO-d₆, 400 MHz): δ1.85-1.91 (m,2H), 2.10 (s, 3H), 2.37 (s, 3H), 3.12 (m, 2H), 3.48 (t, J=6.0 Hz, 2H),5.05 (s, 2H), 6.57 (s, 1H), 7.46 (s, 1H), 7.80 (s, 1H). LC/MS; [M+H]calculated for C₁₃H₁₇N₅O₂; expected 276.14; found 276.10.

Example 9-5b1-(3-chloropropyl)-3-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)urea

1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-amine (342 mg, 1.78mmol) and 2-chloropropyl isocyanate (213 mg, 1.78 mmol) in acetonitrile(5 mL) were heated at 65° C. for 16 hours. The reaction was cooled downto room temperature, concentrated and the residue was dissolved indichloromethane (5 mL) and purified by silica column chromotography(100% to 90% dichloromethane in methanol: 30 minute gradient). The purefractions were combined and concentrated, then triturated with ethylacetate/hexanes (1/9) to afford(1-(3-chloropropyl)-3-(1-((3,5-dimethylisoxazol-4-yl)methyl)1H-pyrazol-4-yl)urea(218 mg, 39%) as white solid. LC/MS; [M+H] calculated for C₁₂H₁₆ClN₅O₂;expected 298.10; found 298.10.

Example 9-61-benzyl-3-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-1,3,5-triazinan-2-one

1-benzyl-5-(2,4-dimethoxybenzyl)-3-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-1,3,5-triazinan-2-one(example 9-6a) (44 mg, 0.09 mmol), anisole (9 mg, 0.09 mmol), and 50%trifluoroacetic acid/dichloromethane solution (1 mL) in dichloromethane(1 mL) were stirred at room temperature for 2 hours. The reaction wasconcentrated, quenched with saturated sodium bicarbonate (50 mL),extracted with ethyl acetate (2×, 50 mL) and washed with brine (50 mL).The combined organic extracts were dried over magnesium sulfate,filtered and concentrated. Purification by silica column chromotography(100% to 90% dichloromethane in methanol: 30 minute gradient) afforded1-benzyl-3-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-1,3,5-triazinan-2-one(19 mg, 62%) as a white solid. LC/MS; [M+H] calculated for C₁₉H₂₂N₆O₂;expected 367.18; found 367.20. The title compound was shown to inhibithT2R08 bitter receptor and had an IC₅₀ of 5.84 μM.

Example 9-6a1-benzyl-5-(2,4-dimethoxybenzyl)-3-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-1,3,5-triazinan-2-one

1-benzyl-3-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)urea(example 9-6b) (50 mg, 0.15 mmol), 2,4-methoxy benzyl amine (26 mg, 0.15mmol), and formaldehyde (37% wt. in water) (25 mg, 0.31 mmol) wereheated at 100° C. for 16 hours. The reaction was cooled to roomtemperature, diluted with brine (50 mL) and extracted withdichloromethane (2×, 50 mL). The combined organic extracts were driedover magnesium sulfate, filtered and concentrated. The residue waspurified by silica column chromotography (100% to 90% dichloromethane inmethanol: 30 minute gradient) to afford1-benzyl-5-(2,4-dimethoxybenzyl)-3-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-1,3,5-triazinan-2-one(44 mg, 56%) as an oil. LC/MS; [M+H] calculated for C₂₈H₃₂N₆O₄; expected517.25; found 517.20.

Example 9-6b1-benzyl-3-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)urea

1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-amine (287 mg, 1.49mmol) and benzyl isocyanate (199 mg, 1.49 mmol) in acetonitrile (5 mL)were heated at 65° C. for 16 hours. The reaction was cooled down to roomtemperature and concentrated. The residue was purified by silica columnchromotography (100% to 90% dichloromethane in methanol: 30 minutegradient) and triturated with ethyl acetate/hexanes (1/9) to afford1-benzyl-3-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)urea(246 mg, 51%) as a white solid. LC/MS; [M+H] calculated for C₁₇H₁₉N₅O₂;expected 326.15; found 326.10.

Example 9-75-benzyl-1-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-3-phenethyl-1,3,5-triazinan-2-one

Prepared as in example 9-6a from1-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-3-phenethylurea(example 9-7a), formaldehyde, and benzyl amine. Yield: 15%. ¹H NMR(DMSO-d₆, 400 MHz): δ2.11 (s, 3H), 2.37 (s, 3H), 2.69 (t, J=7.6 Hz, 2H),3.39 (t, J=7.8 Hz, 2H), 3.81 (s, 2H), 4.16 (s, 2H), 4.42 (s, 2H), 5.05(s, 2H), 7.17-7.32 (m, 10H), 7.42 (s, 1H), 7.78 (s, 1H). LC/MS; [M+H]calculated for C₂₇H₃₀N₆O₂; expected 471.24; found 471.15. The titlecompound was shown to inhibit hT2R08 bitter receptor and had an IC₅₀ of2.64 μM.

Example 9-7a1-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-3-phenethylurea

Prepared as in example 9-6b from1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-amine and phenethylisocyanate. Yield: 29%. ¹H NMR (DMSO-d₆, 400 MHz): δ2.10 (s, 3H), 2.36(s, 3H), 2.69 (t, J=7.2 Hz, 2H), 3.25 (q, J=7.4 Hz, 2H), 5.02 (s, 2H),6.00 (t, J=5.8 Hz, 1H), 7.16-7.30 (m, 6H), 7.68 (s, 1H), 8.13 (s, 1H).LC/MS; [M+H] calculated for C₁₈H₂₁N₅O₂; expected 340.17; found 340.20.

Example 9-81-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-3-phenethyl-1,3,5-triazinane-2,4,6-trione

1-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-3-phenethylurea(example 9-7a) (100 mg, 0.29 mmol) in THF (2 mL) was cooled to 0° C. andn-chlorocarbonyl isocyanate (93 mg, 0.88 mmol) was slowly added. Afteraddition, the reaction was allowed to warm to room temperature andstirred for 1 hour. The reaction was concentrated and the residue waspurified by silica column chromotography (100% to 90% dichloromethane inmethanol: 30 minute gradient) to afford(1-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-3-phenethyl-1,3,5-triazinane-2,4,6-trione(100 mg, 83%) as a white solid. ¹H NMR (DMSO-d₆, 400 MHz): δ2.13 (s,3H), 2.39 (s, 3H), 2.82 (t, J=5.8 Hz, 2H), 3.88 (t, J=8.0 Hz, 2H), 5.17(s, 2H), 7.19-7.31 (m, 5H), 7.44 (s, 1H), 7.89 (s, 1H), 11.84 (s, 1H).LC/MS; [M+H] calculated for C₂₀H₂₀N₆O₄; expected 409.15; found 409.20.The title compound was shown to inhibit hT2R08 bitter receptor and hadan IC₅₀ of 3.03 μM.

Example 9-91-benzyl-3-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-1,3,5-triazinane-2,4,6-trione

1-benzyl-3-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)urea(example 95b) (100 mg, 0.31 mmol) in THF (2 mL) was cooled to 0° C. andn-chlorocarbonyl isocyanate (97 mg, 0.92 mmol) was slowly added. Afteraddition, the reaction was allowed to warm up to room temperature andstirred for 1 hour. The reaction was concentrated and the residue waspurified by silica column chromotography (100% to 90% dichloromethane inmethanol: 30 minute gradient) to afford1-benzyl-3-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-1,3,5-triazinane-2,4,6-trione(112 mg, 93%) as a white solid. ¹H NMR (DMSO-d₆, 400 MHz): δ2.12 (s,3H), 2.38 (s, 3H), 4.87 (s, 2H), 5.16 (s, 2H), 7.23-7.34 (m, 5H), 7.45(s, 1H), 7.90 (s, 1H), 11.93 (s, 1H). LC/MS; [M+H] calculated forC₁₉H₁₈N₆O₄; expected 395.14; found 395.15. The title compound was shownto inhibit hT2R08 bitter receptor and had an IC₅₀ of 1.14 μM.

Example 10 hT2R8 Antagonists: Making the Compounds of the InventionExample 10-13-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)imidazolidine-2,4-dione

1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazole-4-carbonyl azide(example 10-1a) (6 g, 25.5 mmol) in toluene (100 mL) was refluxed for 1hour and cooled to ambient temperature under an atmosphere of nitrogen.Glycine methyl ester-hydrochloride (3.1 g, 26 mmol) and triethylamine(3.2 g, 32 mmol) was added the mixture was refluxed for 16 hours. Thereaction was cooled and the solvent removed on the rotovap. The solidwas redissolved in ethyl acetate (100 mL) and the organic phase waswashed with 1N HCl solution (2×, 150 mL). The aqueous phase was backextracted with ethyl acetate (2×, 75 mL) and the combined organicextracts were dried over sodium sulfate and concentrated. The resultingsolid was triturated with ethyl acetate/hexanes (1/9) and dried underhigh vacuum to afford3-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)imidazolidine-2,4-dione(5.2 g, 74%) as a white solid. ¹H NMR (DMSO-d₆, 400 MHz): δ 2.19 (s,3H), 2.42 (s, 3H), 4.09 (s, 2H), 5.06 (s, 2H), 5.68 (bs, 1H), 7.90 (s,1H), 8.05 (1H). The title compound was shown to inhibit hT2R08 bitterreceptor and had an IC₅₀ of 1.7 μM.

Example 10-1a1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazole-4-carbonyl azide

Sodium nitrite (450 mg, 6.5 mmol, in H₂O) (10 mL) was added dropwise,over 10 minutes, to a suspension of1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazole-4-carbohydrazide(example 10-1b) (1 g, 4.3 mmol) in 10% aqueous acetic acid (50 mL) andcooled to 0° C. via an ice water bath. The reaction was stirred for anadditional 15 minutes then extracted with ethyl acetate (3×, 75 mL). Thecombined organic extracts were washed with aqueous saturated sodiumcarbonate (100 mL) followed by H₂O (100 mL). The combined organicextracts were dried over sodium sulfate, filtered and concentrated. Thesolid product was triturated with ethyl acetate/hexanes (1/9) and drieden vacuo to afford1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazole-4-carbonyl azide (1 g,93%) as a white solid. ¹H NMR (CDCl₃, 400 MHz): δ2.20 (s, 3H), 2.44 (s,3H), 5.07 (s, 2H), 7.81 (s, 1H), 7.93 (s, 1H).

Example 10-1b1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazole-4-carbohydrazide

Ethyl 1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazole-4-carboxylate(example 10-1c) (6 g, 24 mmol) and hydrazine (7.5 g, 240 mmol) werestirred in EtOH (100 mL) at reflux for 12 hours. The solution wasconcentrated on the rotovap and the solid product was triturated withethyl acetate/hexanes (1/9) and dried under high vacuum to afford1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazole-4-carbohydrazide (5.5g, 97%) as a pure white solid. ¹H NMR (DMSO-d₆, 400 MHz): δ2.11 (s, 3H),2.39 (s, 3H), 5.15 (s, 2H), 7.81 (s, 1H), 8.17 (s, 1H), 9.31 (bs, 1H).

Example 10-1c1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazole-4-carboxylate

Ethyl 1H-pyrazole-4-carboxylate (4.2 g, 30 mmol),4-(chloromethyl)-3,5-dimethylisoxazole (5.1 g, 35 mmol), and cesiumcarbonate (9.8 g, 30 mmol), in DMF (50 mL), were stirred at 80° C. for12 hours. The reaction was cooled to ambient temperature, diluted with0.1 N HCl (150 mL) and extracted with ethyl acetate (3×, 75 mL). Thecombined organic extracts were dried over sodium sulfate andconcentrated on the rotovap. The solid product was triturated with ethylacetate/hexanes(1/9) and collected by filtration to afford ethyl1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazole-4-carboxylate (6 g,80%) as a white solid. ¹H NMR (CDCl₃, 400 MHz): δ1.34 (t, J=7.2 Hz, 3H),2.19 (s, 3H), 2.43 (s, 3H), 4.29 (q, J=7.2 Hz, 2H), 5.06 (s, 2H), 7.77(s, 1H), 7.91 (s, 1H).

Example 10-23-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)imidazolidine-2,4-dione

Prepared as in example 10-1 from (+/−)-phenylalanine methyl esterhydrochloride and1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazole-4-carbonyl azide.Yield: 58%. ¹H NMR (Acetone-d₆, 400 MHz): δ2.17 (s, 3H), 2.43 (s, 3H),3.07 (dd, J=14.4, 6.4 Hz, 1H), 3.20 (dd, J=14, 4.4 Hz, 1H), 4.53 (t,J=4.8 Hz, 1H), 5.18 (s, 2H), 7.27-7.19 (m, 5H), 7.46 (bs, 1H), 7.79 (s,1H), 7.99 (s, 1H). MS M+H calculated 366.15; found 366.1. Melting point:169-171° C. The title compound was shown to inhibit hT2R08 bitterreceptor and had an IC₅₀ of 0.18 μM.

Example 10-3(S)-5-benzyl-3-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)imidazolidine-2,4-dione

Prepared as in example 10-1 from (S)-phenylalanine methyl esterhydrochloride and1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazole-4-carbonyl azide(example 6a). Yield: 13% isolated from chiral chromotography ¹H NMR(CDCl₃, 400 MHz): δ2.19 (s, 3H), 2.42 (s, 3H), 2.88 (dd, J=13.6, 9.2 Hz,1H), 3.35 (dd, J=13.6, 3.6 Hz, 1H), 4.31-4.35 (m, 1H) 5.06 (s, 2H), 5.53(bs, 1H), 7.21-7.36 (m, 5H), 7.85 (s, 1H), 8.01 (s, 1H). LC/MS; [M+H]calculated for C₁₉H₁₉N₅O₃; expected 366.15; found 366.1.[α]_(D)=(−)-136, c=0.1, ethanol. The title compound was shown to inhibithT2R08 bitter receptor and had an IC₅₀ of 0.12 μM.

Example 10-4(R)-5-benzyl-3-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)imidazolidine-2,4-dione

Prepared as in example 10-1 from (R)-phenylalanine methyl esterhydrochloride and1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazole-4-carbonyl azide.Yield: 9% isolated from chiral chromotography ¹H NMR (CDCl₃, 400 MHz):δ2.19 (s, 3H), 2.42 (s, 3H), 2.88 (dd, J=13.6, 9.2 Hz, 1H), 3.35 (dd,J=13.6, 3.6 Hz, 1H), 4.31-4.35 (m, 1H) 5.06 (s, 2H), 5.53 (bs, 1H),7.21-7.36 (m, 5H), 7.85 (s, 1H), 8.01 (s, 1H). MS M+H calculated 366.15;found 366.1. [α]_(D)=(+)-124, c=0.2, ethanol. The title compound wasshown to inhibit hT2R08 bitter receptor and had an IC₅₀ of 0.11 μM.

Example 10-53-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-1-(2-phenoxyethyl)imidazolidine-2,4-dione

3-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)imidazolidine-2,4-dione(example 10-1) (200 mg, 0.7 mmol), (2-bromoethoxy)benzene (200 mg, 1mmol), and cesium carbonate (325 mg, 1 mmol) were irradiated in themicrowave reactor at 85° C. for 20 minutes. The reaction was cooled toroom temperature, diluted with aqueous 1N HCl (100 mL) and extractedwith ethyl acetate (3×, 75 mL). The combined organic extracts were driedover sodium sulfate, filtered and concentrated. The residue was taken upin methanol (10 mL) and purified by reversed phase HPLC (5 to 95%acetonitrile in H₂O: 25 minute gradient). The pure fractions werepooled, concentrated then re-dissolved in absolute ethanol andconcentrated on the rotovap (4×) to afford3-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-1-(2-phenoxyethyl)imidazolidine-2,4-dione(150 mg, 54%) as a white solid. ¹H NMR (CDCl₃2.18, 400 MHz): δ (s, 3H),2.41 (s, 3H), 3.86 (t, J=5.2 Hz, 2H), 4.19 (t, J=4.4 Hz, 2H), 4.25 (s,2H), 5.05 (s, 2H), 6.88 (dd, J=9.2, 1.2 Hz, 2H), 7.00 (dt, J=7.6, 1.2Hz, 1H), 7.27-7.32 (m, 2H), 7.89 (s, 1H), 8.05 (s, 1H). MS M+Hcalculated 396.17; found 396.1. Melting point: 117-118° C. The titlecompound was shown to inhibit hT2R08 bitter receptor and had an IC₅₀ of0.06 μM.

Example 10-63-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-1-(3-methoxybenzyl)imidazolidine-2,4-dione

Prepared as in example 10-5 from3-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)imidazolidine-2,4-dione(example 6) and 3-methoxy-benzyl bromide. Yield: 55%. ¹NMR (CDCl₃, 400MHz): δ2.19 (s, 3H), 2.42 (s, 3H), 3.81 (s, 3H), 3.85 (s, 2H), 4.58 (s,2H), 5.06 (s, 2H), 6.81-6.88 (m, 3H), 7.26-7.31 (m, 1H), 7.92 (s, 1H),8.08 (s, 1H). The title compound was shown to inhibit hT2R08 bitterreceptor and had an IC₅₀ of 0.07 μM.

Example 10-7 Methyl3-((3-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-2,4-dioxoimidazolidin-1-yl)methyl)benzoate

Prepared as in example 10-5 from3-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)imidazolidine-2,4-dioneand 3-methoxy-benzyl bromide. Yield: 83%. ¹H NMR (CDCl₃, 400 MHz): δ2.20(s, 3H), 2.43 (s, 3H), 3.86 (s, 2H), 3.93 (s, 3H), 4.67 (s, 2H), 5.07(s, 2H), 7.45-7.52 (m, 2H), 7.93 (s, 1H), 7.95 (s, 1H), 8.03 (dd, J=7.2,1.6 Hz, 1H), 8.08 (s, 1H). The title compound was shown to inhibithT2R08 bitter receptor and had an IC₅₀ of 0.09 μM.

Example 10-83-((3-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-2,4-dioxoimidazolidin-1-yl)methyl)benzoicacid

3-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)imidazolidine-2,4-dione(500 mg, 1.8 mmol) (example 10-5), methyl 3-(bromomethyl)benzoate (456mg, 2 mmol), and cesium carbonate (650 mg, 2 mmol) were stirred in DMF(4 mL) in the microwave reactor at 85° C. for 20 minutes. The reactionwas cooled, diluted with 1N aqueous HCl (100 mL) and extracted withethyl acetate (3×, 75 mL). The combined organic extracts were dried oversodium sulfate, filtered and concentrated. The crude ester was dissolvedin methanol (5 mL) and aqueous NaOH (50 mL, 10% by wt) was added and themixture was stirred at ambient temperature for 2 hours. The reaction wasacidified with 1N HCl (150 mL) and extracted with ethyl acetate (3×, 75mL). The combined organic extracts were dried over sodium sulfate,filtered, and concentrated on the rotovap. The free acid was trituratedwith ethyl acetate/hexanes (1/9) and dried under vacuum to afford3-((3-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-2,4-dioxoimidazolidin-1-yl)methyl)benzoicacid (610 mg, 83%) as a white solid. ¹H NMR (DMSO-d₆, 400 2.13 (s, 3H),2.29 (s, 3H), 4.00 (s, 2H), 4.59 (s, 2H), 5.18 (s, 2H), MHz): δ7.46-7.59 (m, 2H), 7.78 (s, 1H), 7.85-7.88 (m, 2H), 8.18 (s, 1H). Thetitle compound was shown to inhibit hT2R08 bitter receptor and had anIC₅₀ of 1.8 μM.

Example 10-93-((3-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-2,4-dioxoimidazolidin-1-yl)methyl)-N-methylbenzamide

3-((3-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-2,4-dioxoimidazolidin-1-yl)methyl)benzoicacid (100 mg, 0.24 mmol) (example 10-8), methylamine hydrochloride (67mg, 1 mmol), triethylamine (155 mg, 1.5 mmol), and EDC (57 mg, 0.3 mmol)in acetonitrile (3 mL) were irradiated in the microwave reactor at 80°C. for 10 minutes. The reaction was cooled, diluted with aqueous 1N HCl(100 mL) and extracted with ethyl acetate (3×, 75 mL). The combinedorganic extracts were dried over sodium sulfate, filtered andconcentrated. The crude product was dissolved in MeOH (3 mL) andpurified by reversed phase HPLC (5 to 95% acetonitrile in H2O: 25 minutegradient) to afford3-((3-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-2,4-dioxoimidazolidin-1-yl)methyl)-N-methylbenzamide(25 mg, 25%) as white solid. MS M+H calculated 423.17; found 423.2. Thetitle compound was shown to inhibit hT2R08 bitter receptor and had anIC₅₀ of 0.14 μM.

Example 10-103-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-1-(3-hydroxybenzyl)imidazolidine-2,4-dione

Prepared as in example 10-1 from1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazole-4-carbonyl azide(example 10-1a) and methyl 2-(3-hydroxybenzylamino)acetate (example10-10a). Yield: 24%. ¹H NMR (DMSO, 400 MHz): δ2.15 (s, 3H), 2.41 (s,3H), 3.99 (s, 2H), 4.45 (s, 2H), 5.21 (s, 2H), 6.70 (m, 3H), 7.15 (m,H), 7.80 (s, 1H), 8.19 (s, H), 9.44 (s, H). LC/MS; [M+H] expected 382.1;found 382.1. Melting point: 35-136° C. The title compound was shown toinhibit hT2R08 bitter receptor and had an IC₅₀ of 0.035 uM.

Example 10-10a methyl 2-(3-hydroxybenzylamino)acetate

Glycine methyl ester (500 mg, 4 mmol) and 3-hydroxybenzaldehyde (480 mg,4 mmol) were dissolved in 5 mL THF/Methanol (1:1). Acetic acid (240 mg,4 mmol), and 1M sodium cyanoborohydride in THF (4.8 mL, 4.8 mmol) wereslowly added into the reaction. The reaction was irradiated in themicrowave reactor at 85° C. for 15 minutes, cooled to room temperatureand the salts were removed by filtration. The clear solution wasconcentrated and the residue was purified by reversed phase HPLC (10 to95% Acetonitrile in H₂O:25 minute gradient) to give the title compoundas a clear gel. Yield 45%. MS M+H calculated 196.1; found 196.1.

Alternatively3-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-1-(3-hydroxybenzyl)imidazolidine-2,4-dionecan be prepared in large scale as in Example 12-1 from1-(3-(tert-butyldimethylsilyloxy)benzyl)-3-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)imidazolidine-2,4-dione(Example 10-10b, below):

1-(3-(tert-butyldimethylsilyloxy)benzyl)-3-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)imidazolidine-2,4-dione(Example 10-10b) (6.00 g, 12.11 mmol) was dissolved in methanol (100 mL)and treated with HCl (2.0 M solution in diethyl ether, 5 equivalents).The solution was refluxed for 2. The mixture was allowed to cool down toroom temperature and the solvent was removed. The residue was trituratedin toluene, filtered and recrystallized from ethanol at 4° C. to give3-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-1-(3-hydroxybenzyl)imidazolidine-2,4-dione(4.50 g, 11.80 mmol, 97%) as a white solid. ¹H NMR (DMSO, 400 MHz):

2.15 (s, 3H), 2.39 (s, 3H), 3.97 (s, 2H), 4.43 (s, 2H), 5.18 (s, 2H),6.68 (m, 3H), 7.12 (m, 1H), 7.80 (s, 1H), 8.17 (s, H), 9.41 (s, H). MS382 (MH⁺).

Example 10-10b1-(3-(tert-butyldimethylsilyloxy)benzyl)-3-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)imidazolidine-2,4-dione

1-((3,5-dimethylisoxazol-4-yl) methyl)-1H-pyrazole-4-carbonyl azide(Example 10-1a) (3.00 g, 12.20 mmol) along with molecular sieves arestirred at reflux in dry toluene (300 mL) under nitrogen gas until noemission of nitrogen was observed, which indicates complete conversionof 1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazole-4-carbonyl azideinto 4-((4-isocyanato-1H-pyrazol-1-yl)methyl)-3,5-dimethylisoxazole. Asolution of methyl 2-(3-(tert-butyldimethylsilyloxy)benzylamino)acetate(Example 10-10c) (3.78 g, 12.20 mmol) in dry toluene (50 mL) wasprepared and added in one portion at 50° C. to the reaction mixture. Thereaction was refluxed over the weekend, then cooled down to roomtemperature and filtered over a pad of celite. The filtrate wasconcentrated and to give1-(3-(tert-butyldimethylsilyloxy)benzyl)-3-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)imidazolidine-2,4-dioneas a yellowish oily used in the next step without further purification.MS 496 (MH⁺). ¹H NMR (CDCl₃, 400 MHz): δ 0.19 (s, 6H), 0.97 (s, 9H),2.19 (s, 3H), 2.42 (s, 3H), 3.83 (s, 2H), 4.55 (s, 2H), 5.06 (s, 2H),6.75 (m, 1H), 6.80 (m, 1H), 6.86 (m, 1H), 7.23 (t, J=8.0 Hz, 1H).

Example 10-10c methyl2-(3-(tert-butyldimethylsilyloxy)benzylamino)acetate

3-Hydroxybenzaldehyde (10 g, 81.88 mmol) was dissolved in anhydrous THF(200 mL) and N-ethyl-N-isopropylpropan-2-amine (Huenig's base, 31.8 mL,˜3 equivalent) was added. Upon addition of Hunig's base, the solutionchanged to yellow and was cooled down to 0° C. A solution oftert-butylchlorodimethylsilane (13.6 g, 1.1 equiv.) in THF (100 mL) wasadded dropwise. After stirring for 60 hours at room temperature, methyl2-aminoacetate hydrochloride (21 g, 16.73 mmol, ˜2 equiv.) was added andthe mixture was stirred for 6 hours at room temperature. The reactionmixture was cooled down to about 10° C. using ice water and whilestirring vigorously, it was treated rapidly with NaBH(OAc)₃ (35 g,16.51, ˜2 equiv.). After addition was completed, the cooling wasimmediately removed and the reaction mixture was stirred overnight undernitrogen. The mixture was quenched with saturated aqueous NaHCO₃solution and the layers were separated. The aqueous phase was furtherextracted with DCM (2×300 mL). The combined organic extract was washedwith water (50 mL), brine (100 mL) and dried over MgSO₄. The solvent wasremoved under vacuum and the residue purified over silica gel using 25%ethyl acetate in hexanes. After evaporation of solvent from cleanfractions methyl 2-(3-(tert-butyldimethylsilyloxy)benzylamino)acetate(19.9 g, 64.30 mmol, 78%) was obtained as a colorless liquid. NMR(DMSO-d₆, 400 MHz): δ 0.17 (s, 6H), 0.94 (s, 9H), 2.47 (br s, 1H), 3.28(s, 2H), 3.61 (s, 3H), 3.65 (s, 2H), 6.70 (ddd, J=0.8, 1.2, 8.0 Hz, 1H),6.81 (m, 1H), 6.89 (br dt, J=0.8, 7.6 Hz, 1H), 7.17 (pseudo t, J=7.6,8.0 Hz, 1H).

Example 10-113-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-1-(2-hydroxybenzyl)imidazolidine-2,4-dione

Prepared as in example 10-1 from1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazole-4-carbonyl azide(example 10-1a) and methyl 2-(2-hydroxybenzylamino)acetate (example10-11a). Yield: 28%. ¹H NMR (DMSO, 400 MHz): δ2.12 (s, 3H), 2.38 (s,3H), 4.00 (s, 2H), 4.45 (s, 2H), 5.17 (s, 2H), 6.83 (m, 2H), 7.10 (m,2H), 7.78 (s, 1H), 8.16 (s, H), 9.66 (s, H). LC/MS; [M+H] expected382.1; found 382.2. The title compound was shown to inhibit hT2R08bitter receptor and had an IC₅₀ of 0.07 uM.

Example 10-11a methyl 2-(2-hydroxybenzylamino)acetate

Prepared as in example 10-10a from glycine methyl ester and2-hydroxybenzaldehyde. Yield 40%. MS M+H calculated 196.1; found 196.1

Example 10-123-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-1-(4-hydroxybenzyl)imidazolidine-2,4-dione

Prepared as in example 10-1 from1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazole-4-carbonyl azide andmethyl 2-(4-hydroxybenzylamino)acetate (example 10-12a). Yield 9%. ¹HNMR (DMSO, 400 MHz): δ2.117 (s, 3H), 2.383 (s, 3H), 3.918 (s, 2H), 4.387(s, 2H), 5.174 (s, 2H), 6.719 (J=8.8, d, 2H), 7.108 (J=8.8, m, 2H),7.761 (s, 1H), 8.154 (s, H), 9.399 (s, H). LC/MS; [M+H] expected 382.1;found 382.2. The title compound was shown to inhibit hT2R08 bitterreceptor and had an IC₅₀ of 0.06 uM.

Example 10-12a methyl 2-(4-hydroxybenzylamino)acetate

Prepared as in example 10-10a from glycine methyl ester and4-hydroxybenzaldehyde. Yield 40%. MS M+H calculated 196.1; found 196.1

Example 10-133-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-1-(3-hydroxy-4-methoxybenzyl)imidazolidine-2,4-dione

Prepared as in example 10-1 from1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazole-4-carbonyl azide(example 10-1a) and methyl 2-(3-hydroxy-4-methoxybenzylamino)acetate(example 10-13a). Yield 22%. ¹H NMR (DMSO, 400 MHz): δ2.119 (s, 3H),2.383 (s, 3H), 3.716 (s, 3H), 3.923 (s, 2H), 4.361 (s, 2H), 5.117 (s,2H), 6.667 (m, 2H), 6.863 (J=8.4, d, 1H), 7.766 (s, H), 8.159 (s, H). MSM+H calculated 412.1; found 412.1. The title compound was shown toinhibit hT2R08 bitter receptor and had an IC₅₀ of 0.1 uM.

Example 10-13a methyl 2-(3-hydroxy-4-methoxybenzylamino)acetate

Prepared as in example 10-10a from glycine methyl ester and3-hydroxy-4-methoxybenzaldehyde. Yield 47%. MS M+H calculated 226.1;found 226.1

Example 10-143-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-1-(3-(2-methoxyethoxy)benzyl)imidazolidine-2,4-dione

Prepared as in example 10-1 from1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazole-4-carbonyl azide(example 10-1a) and methyl 2-(3-(2-methoxyethoxy)benzylamino)acetate(example 10-14a). Yield 27%. ¹H NMR (DMSO, 400 MHz): δ2.12 (s, 3H), 2.38(s, 3H), 3.26 (s, 3H), 3.62 (t, J=4.4, 2H), 3.98 (s, 2H), 4.06 (t,J=4.4, 2H), 4.48 (s, 2H), 5.18 (s, 2H), 6.86 (m, 3H), 7.24 (t, J=8, 1H),7.78 (s, 1H), 8.17 (s, 1H). MS M+H calculated 440.2; found 440.2. Thetitle compound was shown to inhibit hT2R08 bitter receptor and had anIC₅₀ of 0.07 uM.

Example 10-14a methyl 2-(3-(2-methoxyethoxy)benzylamino)acetate

Prepared as in example 10-10a from glycine methyl ester and3-(2-methoxyethoxy)benzaldehyde. Yield 55%. MS M+H calculated 254.1;found 254.1

Example 10-153-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-1-(2-(methylthio)benzyl)imidazolidine-2,4-dione

Prepared as in example 10-1 from1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazole-4-carbonyl azide(example 10-1a) and methyl 2-(2-(methylthio)benzylamino)acetate (example10-15a). Yield 67%. ¹H NMR (DMSO, 400 MHz): δ2.12 (s, 3H), 2.39 (s, 3H),2.48 (s, 3H), 3.98 (s, 2H), 4.54 (s, 2H), 5.19 (s, 2H), 7.18 (m, 1H),7.30 (m, 3H), 7.79 (s, 1H), 8.18 (s, 1H). LC/MS; [M+H] expected 412.1;found 412.1. The title compound was shown to inhibit hT2R08 bitterreceptor and had an IC₅₀ of 0.03 uM.

Example 10-15a methyl 2-(2-(methylthio)benzylamino)acetate

Prepared as in example 10-10a from glycine methyl ester and2-(methylthio)benzaldehyde. Yield 50%. MS M+H calculated 226.1; found226.1

Example 10-163-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-1-(2-(2-methoxyethoxy)benzyl)imidazolidine-2,4-dione

Prepared as in example 10-5 from3-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-1-(2-hydroxybenzyl)imidazolidine-2,4-dione(example 10-11) and 2-bromoethyl methyl ether. Yield 19%. ¹H NMR (DMSO,400 MHz): δ2.11 (s, 3H), 2.08 (s, 3H), 3.25 (s, 3H), 3.64 (t, J=3.6,2H), 4.00 (s, 2H), 4.11 (t, J=3.2, 2H), 4.27 (s, 2H), 5.17 (s, 2H), 6.90(m, 1H), 7.00 (m, 1H), 7.26 (m, 2H), 7.76 (s, 1H), 8.15 (s, 1H). Thetitle compound was shown to inhibit hT2R08 bitter receptor and had anIC₅₀ of 0.1 uM.

Example 10-1733-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-1-(2-(methylsulfinyl)benzyl)imidazolidine-2,4-dione

In a 20 mL microwave vial,3-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-1-(2-(methylthio)benzyl)imidazolidine-2,4-dione(example 10-15) (70 mg, 0.17 mmol) and m-CPBA (58 mg, 0.34 mmol) weredissolved in dichloromethane at 0° C. The reaction was stirred at 0° C.and allowed to warm up to room temperature for 4 hours. Solvent of thereaction was removed under vacuum, and the crude product was dissolvedin 1 mL ethanol and was purified by varian HPLC (10 to 95%Acetonitrile/water; 25 minutes). The purified fraction was evaporatedunder vacuum to give the title compound. MS M+H calculated 428.1; found428.1. The title compound was shown to inhibit hT2R08 bitter receptorand had an IC₅₀ of 0.4 uM. Yield: 12 mg, 17%.

Example 10-183-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-1-(2-methoxybenzyl)imidazolidine-2,4-dione

Prepared as in example 10-5 from3-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)imidazolidine-2,4-dione(example 10-1) and 1-(bromomethyl)-2-methoxybenzene (199 mg, 1 mmol).Yield: 33%. MS M+H calculated 396.1; found 396.1. The title compound wasshown to inhibit hT2R08 bitter receptor and had an IC₅₀ of 0.06 uM.

Example 10-192-((3-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-2,4-dioxoimidazolidin-1-yl)methyl)benzonitrile

Prepared as in example 10-5 from3-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)imidazolidine-2,4-dione(example 10-1) and 2-(bromomethyl)benzonitrile. Yield: 27%. MS M+Hcalculated 391.1; found 391.1. The title compound was shown to inhibithT2R08 bitter receptor and had an IC₅₀ of 0.5 μM.

Example 10-203-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-1-(2-methylbenzyl)imidazolidine-2,4-dione

Prepared as in example 10-5 from3-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)imidazolidine-2,4-dione(example 10-1) and 1-(bromomethyl)-2-methylbenzene. Yield: 21%. MS M+Hcalculated 380.1; found 380.1. The title compound was shown to inhibithT2R08 bitter receptor and had an IC₅₀ of 0.1 uM.

Example 10-213-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-1-(2-fluorobenzyl)imidazolidine-2,4-dione

Prepared as in example 10-5 from3-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)imidazolidine-2,4-dione(example 10-1) and 1-(bromomethyl)-2-fluorobenzene. Yield 42%. MS M+Hcalculated 384.1; found 384.1. The title compound was shown to inhibithT2R08 bitter receptor and had an IC₅₀ of 0.08 uM.

Example 10-223-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-1-(2-(trifluoromethyl)benzyl)imidazolidine-2,4-dione

Prepared as in example 10-5 from3-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)imidazolidine-2,4-dione(example 10-1) and 1-(bromomethyl)-2-(trifluoromethyl)benzene. Yield:37%. MS M+H calculated 434.1; found 434.1. The title compound was shownto inhibit hT2R08 bitter receptor and had an IC₅₀ of 0.2 uM.

Example 10-233-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-1-(2-nitrobenzyl)imidazolidine-2,4-dione

Prepared as in example 10-5 from3-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)imidazolidine-2,4-dione(example 10-1) and 1-(bromomethyl)-2-nitrobenzene. Yield 22%. MS M+Hcalculated 411.1; found 411.1. The title compound was shown to inhibithT2R08 bitter receptor and had an IC₅₀ of 0.07 uM.

Example 10-243-((3-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-2,4-dioxoimidazolidin-1-yl)methyl)benzaldehyde

Prepared as in example 10-5 from3-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)imidazolidine-2,4-dione(example 10-1) and 3-(bromomethyl)benzaldehyde. Yield: 35%. ¹H NMR(DMSO, 400 MHz): δ2.123 (s, 3H), 2.388 (s, 3H), 4.035 (s, 2H), 4.631 (s,2H), 5.186 (s, 2H), 7.581 (m, 1H), 7.643 (m, 1H), 7.787 (m, 3H), 8.178(s, H), 9.997 (s, H). The title compound was shown to inhibit hT2R08bitter receptor and had an IC₅₀ of 0.2 uM.

Example 10-253-((3-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-2,4-dioxoimidazolidin-1-yl)methyl)benzonitrile

Prepared as in example 10-5 from3-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)imidazolidine-2,4-dione(example 10-1) and 3-(bromomethyl)benzonitrile. Yield 21%. MS M+Hcalculated 411.1; found 411.1. The title compound was shown to inhibithT2R08 bitter receptor and had an IC₅₀ of 1 uM.

Example 10-263-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-1-(3-methylbenzyl)imidazolidine-2,4-dione

Prepared as in example 10-5 from3-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)imidazolidine-2,4-dione(example 10-1) and 1-(bromomethyl)-3-methylbenzene. Yield 25%. MS M+Hcalculated 380.1; found 380.1. The title compound was shown to inhibithT2R08 bitter receptor and had an IC₅₀ of 0.02 uM.

Example 10-273-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-1-(3-fluorobenzyl)imidazolidine-2,4-dione

Prepared as in example 10-5 from3-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)imidazolidine-2,4-dione(example 10-1) and 1-(bromomethyl)-3-fluorobenzene. Yield 27%. MS M+Hcalculated 384.1; found 384.1. The title compound was shown to inhibithT2R08 bitter receptor and had an IC₅₀ of 0.06 uM.

Example 10-281-((1,5-dimethyl-1H-pyrazol-3-yl)methyl)-3-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)imidazolidine-2,4-dione

Prepared as in example 10-5 from3-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)imidazolidine-2,4-dione(example 10-1) and 3-(bromomethyl)-1,5-dimethyl-1H-pyrazole. Yield: 22%.MS M+H calculated 384.1; found 384.1. The title compound was shown toinhibit hT2R08 bitter receptor and had an IC₅₀ of 0.3 uM

Example 10-293-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-1-(4-methoxybenzyl)imidazolidine-2,4-dione

Prepared as in example 10-5 from3-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)imidazolidine-2,4-dione(example 10-1) and 1-(bromomethyl)-4-methoxybenzene. Yield 19%. MS M+Hcalculated 396.1; found 396.1. The title compound was shown to inhibithT2R08 bitter receptor and had an IC₅₀ of 0.07 uM.

Example 10-303-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-1-(4-methylbenzyl)imidazolidine-2,4-dione

Prepared as in example 10-5 from3-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)imidazolidine-2,4-dione(example 10-1) and 1-(bromomethyl)-4-methylbenzene. Yields 25%. MS M+Hcalculated 380.1; found 380.1. The title compound was shown to inhibithT2R08 bitter receptor and had an IC₅₀ of 0.06 uM.

Example 10-313-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-1-(4-fluorobenzyl)imidazolidine-2,4-dione

Prepared as in example 10-5 from3-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)imidazolidine-2,4-dione(example 10-1) and 1-(bromomethyl)-4-fluorobenzene. Yield 33%. MS M+Hcalculated 384.1; found 384.1. The title compound was shown to inhibithT2R08 bitter receptor and had an I IC₅₀ of 0.05 uM.

Example 10-324-((3-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-2,4-dioxoimidazolidin-1-yl)methyl)benzonitrile

Prepared as in example 10-5 from3-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)imidazolidine-2,4-dione(example 10-1) and 4-(bromomethyl)benzonitrile. Yield 21%. MS M+Hcalculated 391.1; found 391.2. The title compound was shown to inhibithT2R08 bitter receptor and had an IC₅₀ of 0.05 uM.

Example 10-333-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-1-(3-(hydroxymethyl)benzyl)imidazolidine-2,4-dione

3-((3-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-2,4-dioxoimidazolidin-1-yl)methyl)benzaldehyde(example 10-24) (131 mg, 0.3 mmol) was dissolved in 2 mL ethanol. Thesolution was passed through the H-Cube instrument at room temperatureusing 10% Pd/C catalyst at a flow rate of 1 ml/minute. The collectedfraction was concentrated, redissolved in 2 mL ethanol and purified byHPLC (10-95% Acetonitrile/Water, 25 minutes). The purified fractionswere combined and concentrated to give the title compound. ¹H NMR (DMSO,400 MHz): δ2.123 (s, 3H), 2.388 (s, 3H), 3.978 (s, 2H), 4.516 (s, 2H),5.182 (s, 2H), 7.242 (m, 4H), 7.779 (s, 1H), 8.172 (s, 1H), 8.505 (s,1H). MS M+H calculated 396.1; found 396.1. The title compound was shownto inhibit hT2R08 bitter receptor and had an IC₅₀ of 0.3 uM. Yield: 24mg, 18%.

Example 10-341-(2-aminobenzyl)-3-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)imidazolidine-2,4-dione

3-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-1-(2-nitrobenzyl)imidazolidine-2,4-dione(example 10-23) (126 mg, 0.3 mmol) was dissolved in 2 mL ethanol. Thesolution was passed through the H-Cube instrument at room temperatureusing 10% Pd/C catalyst at a flow rate of 1 ml/minute. The collectedfraction was concentrated, redissolved in 2 mL ethanol and purified byHPLC (10-95% Acetonitrile/Water, 25 minutes). The purified fractionswere combined and concentrated to afford the title compound. Yield 26%.MS M+H calculated 381.1; found 381.1. The title compound was shown toinhibit hT2R08 bitter receptor and had an IC₅₀ of 0.02 uM.

Example 10-351-(3,4-dimethoxybenzyl)-3-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)imidazolidine-2,4-dione

3-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)imidazolidine-2,4-dione(example 10-1) (275 mg, 1 mmol), (3,4-dimethoxyphenyl)methanol (201 mg,1.2 mmol), N,N,N,N-tetramethylazodicarboxamide (344 mg, 2 mmol) weredissolved in 2 mL anhydrous THF. Tributylphosphine (404 mg, 2 mmol) wasadded and the reaction mixture was placed in a microwave reactor for 5minutes at 90° C. The reaction was filtered, concentrated and purifiedby HPLC (10-95% Acetonitrile/Water, 25 minutes) to afford the titlecompound. Yield: 25 mg, 6%. ¹NMR (DMSO, 400 MHz): δ2.119 (s, 3H), 2.385(s, 3H), 3.724 (J=6.4 d, 6H), 3.946 (s, 2H), 4.435 (s, 2H), 5.178 (s,2H), 6.885 (m, 3H), 7.776 (s, 1H), 8.166 (s, 1H). MS M+H calculated426.1; found 426.1. The title compound was shown to inhibit hT2R08bitter receptor and had an IC₅₀ of 0.06 uM.

Example 10-361-(benzo[d][1,3]dioxol-5-ylmethyl)-3-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)imidazolidine-2,4-dione

Prepared as in example 10-35 from3-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)imidazolidine-2,4-dione(example 10-1) and benzo[d][1,3]dioxol-5-ylmethanol. Yield: 19%. ¹H NMR(DMSO, 400 MHz): δ2.143 (s, 3H), 2.408 (s, 3H), 3.977 (s, 2H), 4.440 (s,2H), 5.202 (s, 2H), 6.003 (s, 2H), 6.897 (m, 3H), 7.788 (s, 1H), 8.181(s, 1H). MS M+H calculated 410.1; found 410.1. The title compound wasshown to inhibit hT2R08 bitter receptor and had an IC₅₀ of 0.07 uM.

Example 10-371-(3-((dimethylamino)methyl)benzyl)-3-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)imidazolidine-2,4-dione

3-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)imidazolidine-2,4-dione(example 10-1) (275 mg, 1 mmol) 1,3-bis(bromomethyl)benzene (263 mg, 1mmol), and cesium carbonate (325 mg, 1 mmol) were dissolved in 2 mL DMFand irradiated in the microwave reactor at 165° C. for 5 minutes. Thereaction was cooled to room temperature, and salt precipitate wasremoved by filtration. The clear solution containing crude product wasconcentrated and redissolved in ethyl acetate. The organic solution waswashed with water twice followed by brine. The organic layer was driedover sodium sulfate and evaporated to give the crude product which wascarried forward to the next step without further purification orcharacterization.1-(3-(bromomethyl)benzyl)-3-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)imidazolidine-2,4-dione(example 42a) (152 mg, 0.3 mmol), dimethylamine (2 M solution in THF)(1.5 mL, 3 mmol), and sodium hydride (9 mg, 0.36 mmol) were dissolved in1 mL anhydrous THF. The reaction was placed in a microwave reactor for 5minutes at 120° C. The crude product was redissolved in 2 mL ethanol andwas purified by HPLC (10-95% Acetonitrile/Water, 25 minutes) to afford1-(3-((dimethylamino)methyl)benzyl)-3-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)imidazolidine-2,4-dione(16mg, 13%). MS M+H calculated 423.1; found 423.1. The title compound wasshown to inhibit hT2R08 bitter receptor and had an IC₅₀ of 1.2 uM.

Example 10-383-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-1-((1-methyl-1H-pyrazol-3-yl)methyl)imidazolidine-2,4-dione

Prepared as in example 10-5 from3-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)imidazolidine-2,4-dione(example 10-1) and 3-(bromomethyl)-1-methyl-1H-pyrazole. Yield 19%. MSM+H calculated 370.1; found 370. The title compound was shown to inhibithT2R08 bitter receptor and had an IC₅₀ of 0.4 uM.

Example 10-39N-(2-((3,5-dimethylisoxazol-4-yl)methyl)-2H-tetrazol-5-yl)-3-methoxybenzamide

2-((3,5-dimethylisoxazol-4-yl)methyl)-2H-tetrazol-5-amine (Example10-39a) (102 mg, 0.528 mmol), 3-methoxybenzoyl chloride (0.065 mL, 0.528mmol) and pyridine (0.043 mL, 0.528 mmol) in acetonitrile (3 mL) werestirred at 100° C. for one hour. The reaction was diluted withdichloromethane (30 mL) and washed with brine (30 mL). The organics weredried over sodium sulfate, concentrated and purified by reverse phaseHPLC (Solvent system: acetonitrile/water 10% to 100% gradient), 25minute run) affordingN-(2-((3,5-dimethylisoxazol-4-yl)methyl)-2H-tetrazol-5-yl)-3-methoxybenzamideas a white crystalline solid (60 mg, 35% yield) MS M+H calculated 329.1,found 329. ¹H NMR (400 MHz, DMSO-d6): δ 2.02 (s, 3H), 2.46 (s, 3H), 3.81(s, 3H), 5.78 (s, 2H), 7.16 (m, 1H), 7.42 (t, J=8 Hz, 2H), 7.54 (m, 1H),11.3 (s, 1H). The compound had an IC₅₀ on hT2R8 bitter receptor of 1.87μM.

Example 10-39a 2-((3,5-dimethylisoxazol-4-yl)methyl)-2H-tetrazol-5-amine

2H-tetrazol-5-amine (1.29 g, 12.5 mmol),4-(chloromethyl)-3,5-dimethylisoxazole (1.56 mL, 12.5 mmol) andpotassium carbonate (1.73 g, 15.5 mmol) in DMF (20 mL) were heated to80° C. with stirring for 16 hours. The reaction was cooled to roomtemperature, diluted with dichloromethane (100 mL) and washedconsecutively with brine and water. The organics were dried over sodiumsulfate and concentrated with rotary evaporation. The crude product waspurified by silica gel chromatography (0-10% gradient ethylacetate/dichloromethane) affording2-((3,5-dimethylisoxazol-4-yl)methyl)-2H-tetrazol-5-amine as a whitecrystalline solid (970 mg, 40% yield) MS M+H calculated 195.1, found195.

Example 10-40N-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-imidazol-4-yl)benzo[d][1,3]dioxole-5-carboxamide

1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-imidazol-4-amine (example10-40a) (110 mg, 0.57 mmol), benzo[d][1,3]dioxole-5-carbonyl chloride(105 mg, 0.57 mmol), and triethylamine (90 μL, 0.69 mmol) indichloromethane was stirred for 16 hours. The reaction was diluted withdichloromethane (30 mL) and washed consecutively with brine and water.The organics were dried over sodium sulfate and concentrated by rotaryevaporation. The crude product was purified by reverse phase HPLC(Solvent system: acetonitrile/water, 10% to 100% gradient, 25 minuterun) to affordN-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-imidazol-4-yl)benzo[d][1,3]dioxole-5-carboxamideas a white crystalline solid (32 mg, 15% yield). MS M+H calculated341.3, found 341.3. ¹H NMR (400 MHz, DMSO-d6): δ 2.08 (s, 3H), 2.41 (s,3H), 5.02 (s, 2H), 6.07 (s, 2H), 6.95 (d, J=8.4 Hz, 1H), 7.27 (d, J=1.6Hz, 1H), 7.54 (m, 3H), 10.6 (s, 1H). The compound had an IC₅₀ on hT2R8bitter receptor of 12.1 μM.

Example 10-40a 1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-imidazol-4-amine

3,5-dimethyl-4-((4-nitro-1H-imidazol-1-yl)methyl)isoxazole (example10-40b) (1.0 g, 4.5 mmol) and 10% palladium on charcoal (200 mg) inmethanol (40 mL) was shaken on a Parr shaker under a pressure of 2.5 barhydrogen for 2 hours. Filtration through a plug of celite followed byrotary evaporation afforded1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-imidazol-4-amine as ayellowish-red solid (800 mg, 93% yield). MS M+H calculated 193, found193.

Example 10-40b3,5-dimethyl-4-((4-nitro-1H-imidazol-1-yl)methyl)isoxazole

3,5-dimethyl-4-((4-nitro-1H-imidazol-1-yl)methyl)isoxazole was preparedin a similar manner to example 10-41c by alkylation of4-nitro-1H-imidazole affording3,5-dimethyl-4-((4-nitro-1H-imidazol-1-yl)methyl)isoxazole as a whitecrystalline solid (5.0 g, 80% yield). MS M+H calculated 223, found 223.¹H NMR (400 MHz, DMSO-d6): δ, ppm: 2.09 (s, 3H), 2.43 (s, 3H), 5.15 (s,2H), 7.90 (d, J=1.6 Hz, 1H), 8.35 (d, J=1.9 Hz, 1H).

Example 10-413-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-5-methylimidazolidine-2,4-dione

1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-amine hydrochloride(example 10-41a) (1.0 g, 5.20 mmol), ethyl-2-isocynatepropionate (0.745g, 5.20 mmol) and triethylamine (1.5 mL, 10.4 mmol) were mixed in EtOH(20 mL). The reaction was refluxed for 12 hours and then allowed to coolto room temperature. Solvent was removed under vacuum and crystalsformed upon standing. The crystals were collected and washed withhexanes to afford the3-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-5-methylimidazolidine-2,4-dionein 80% yield as a white solid. ¹H NMR (DMSO-d₆, 400 MHz): δ 1.53-1.51(d, 3H), 2.19 (s, 3H), 2.42 (s, 3H), 4.21-4.19 (m, 1H), 5.06 (s, 2H),6.00 (bs, 1H), 7.90 (s, 1H), 8.05 (s, 1H). The title compound was shownto inhibit hT2R08 bitter receptor and had an IC₅₀ of 1.3 μM.

Example 10-41a 1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-aminehydrochloride

Tert-butyl1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-ylcarbamate (example10-41b) (592 mg, 2 mmol) was stirred in a solution of 4N HCl in dioxane(20 mL) at ambient temperature for 2 hours. The solvent was removed andthe residue was dissolved in a 1/1 mixture of ethyl acetate/hexanes (30mL) and concentrated twice. The solid was triturated with hexanes andcollected by filtration providing1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-amine hydrochloride(500 mg, 99%) as a white solid. ¹H NMR (DMSO-d₆, 400 MHz): δ 2.11 (s,3H), 2.38 (s, 3H), 5.16 (s, 2H), 7.51 (s, 1H), 8.03 (s, 1H), 10.27 (bs,3H).

Example 10-41b tert-butyl1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-ylcarbamate

3,5-dimethyl-4-((4-nitro-1H-pyrazol-1-yl)methyl)isoxazole (example10-41c) (12 g, 53.8 mmol) and BOC anhydride (12.8 g, 64 mmol) weredissolved in a 3/1/1 mixture of MeOH/EtOH/THF (300 mL) in a Parrreaction bottle, followed by the addition of 10% Pd/C (1.5 g). Themixture was shaken on the Parr hydrogenator under 2 atmospheres ofhydrogen for 3 hours. The mixture was filtered through a 3 inch plug ofcelite and concentrated on the rotovap. The pink oil was purified bysilica gel chromatography (25% ethyl acetate in hexanes) to affordtert-butyl1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-ylcarbamate (12.6 g,80%) as a pink/red oil which solidified on standing to a light pinksolid. ¹H NMR (CDCl₃, 400 MHz): δ 1.41 (s, 9H), 2.10 (s, 3H), 2.32 (s,3H), 4.90 (s, 2H), 6.19 (bs, 1H), 7.19 (s, 1H), 7.50 (s, 1H).

Example 10-41c 3,5-dimethyl-4-((4-nitro-1H-pyrazol-1-yl)methyl)isoxazole

1H-pyrazole (10 g, 147 mmol) was added in small portions to concentratedH₂SO₄ (100 mL), cooled to 0° C. via an ice/water bath, maintaining theinternal reaction temperature below 40° C. Concentrated HNO₃ (10 mL) wascarefully added, dropwise, to the reaction mixture maintaining theinternal reaction temperature below 55° C. The reaction was then heatedto 55° C. and stirred for 5 hours. The mixture was cooled to 0° C. andcarefully made basic (pH˜8) with aqueous NaOH solution (110 g NaOH in150 mL H₂O) until a white precipitate formed, carefully ensuring theinternal temperature of the solution remain below 40° C. The white solidwas collected by filtration and washed with ethyl acetate/hexanes (1/3)then dried en vacuo to afford 4-nitro-1H-pyrazole (7 g, 42%, isolatedyield). ¹³C NMR (DMSO-d₆, 137.0, 126.4. To 4-nitro-1H-pyrazole (9 g, 80mmol) in DMF (100 mL) 100 MHz) δ was added cesium carbonate (26 g, 80mmol) followed by the addition of 4-(chloromethyl)-3,5-dimethylisoxazole(12.3 g, 85 mmol). The reaction mixture was stirred in DMF (100 mL) at80° C. for 30 minutes, then cooled, diluted with H₂O (150 mL) andextracted with ethyl acetate (3×, 75 mL). The combined organic layerswere dried over sodium sulfate, filtered and concentrated. The residuewas taken up in ethyl acetate (200 mL) and washed with H₂O (2×, 100 mL).The organic layer was dried over sodium sulfate, filtered andconcentrated. The solid product was triturated with ethylacetate/hexanes(1/9) and collected by filtration. The product was driedunder high vacuum to afford3,5-dimethyl-4-((4-nitro-1H-pyrazol-1-yl)methyl)isoxazole (12 g, 67%) asa light yellow solid. ¹H NMR (CDCl₃, 400 MHz): δ 2.23 (s, 3H), 2.46 (s,3H), 5.08 (s, 2H), 8.02 (s, 1H), 8.08 (s, 1H).

Example 10-425-benzyl-3-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-1-methylimidazolidine-2,4-dione

Prepared as in example 10-5 from5-benzyl-3-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)imidazolidine-2,4-dione(example 10-2) and iodomethane. Yield: 95%. ¹H NMR (CDCl₃, 400 MHz): δ2.04 (s, 3H), 2.15 (s, 3H), 2.96 (s, 3H), 3.24-3.23 (m, 2H, J=4.0 Hz),4.23-4.21 (m, 1H), 5.00 (s, 2H), 7.24-7.23 (m, 5H, J=4.0 Hz), 7.70 (s,1H), 7.87 (s, 1H).

The title compound was shown to inhibit hT2R08 bitter receptor and hadan IC₅₀ of 0.15 μM.

Example 10-431-benzyl-3-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-5-methylimidazolidine-2,4-dione

Prepared as in example 10-5 from3-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-5-methylimidazolidine-2,4-dione(example 10-41) and benzyl bromide. Yield: 50%. ¹H NMR (CDCl₃, 400 MHz):δ 1.44 (s, 3H), 2.19 (s, 3H), 3.88 (s, 3H), 4.18 (d, J=8 Hz, 2H), 4.22(t, 1H, J=4 Hz)), 5.06 (s, 2H), 7.39-7.29 (m, 5H), 7.94 (s, 1H), 8.10(s, 1H). The title compound was shown to inhibit hT2R08 bitter receptorand had an IC₅₀ of 0.02 μM.

Example 10-442-(1-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-2,5-dioxoimidazolidin-4-yl)aceticacid

Prepared as in example 10-1 from1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazole-4-carbonyl azide(example 10-1a) and H-Asp-OMe. Yield: 85%. MS M+H calculated 334.1;found 334.1

Example 10-453-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-5-phenethylimidazolidine-2,4-dione

Prepared as in Example 10-1 from1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazole-4-carbonyl azide(example 10-1a) and methyl 2-amino-4-phenylbutanoate. Yield: 15%. ¹H NMR(CDCl₃, 400 MHz): δ 2.09-2.02 (m, 2H), 2.19 (s, 3H), 2.41 (s, 3H),2.83-2.78 (m, 2H), 4.13-4.09 (t, 1H, J=8 Hz), 5.05 (s, 2H), 5.95 (bs,1H), 7.30-7.19 (m, 5H), 7.95 (s, 1H), 8.03 (s, 1H). The title compoundwas shown to inhibit hT2R08 bitter receptor and had an IC₅₀ of 0.22 μM.

Example 10-463-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-5-(3-phenylpropyl)imidazolidine-2,4-dione

Prepared as in Example 10-1 from1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazole-4-carbonyl azide(example 10-1a) and methyl 2-amino-5-phenylpentanoate. Yield: 20%. ¹HNMR (CDCl₃, 400 MHz): δ 1.72-1.68 (m, 1H), 1.85-1.78 (m, 2H), 1.99-1.91(m, 1H), 2.19 (s, 3H), 2.41 (s, 3H), 2.69-2.63 (t, J=8 Hz, 2H), 4.13(bs, 1H), 5.05 (s, 2H), 5.95 (bs, 1H), 7.30-7.19 (m, 5H), 7.95 (s, 1H),8.03 (s, 1H). The title compound was shown to inhibit hT2R08 bitterreceptor and had an IC₅₀ of 0.17 μM.

Example 10-475-(benzyloxymethyl)-3-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)imidazolidine-2,4-dione

Prepared as in Example 10-1 from1-(3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazole-4-carbonyl azide(example 10-1a) and methyl 2-amino-3-(benzyloxy)propanoate. Yield: 32%.¹H NMR (CDCl₃, 400 MHz): δ 2.19 (s, 3H), 2.43 (s, 3H), 3.70-3.67 (m,1H), 3.89-3.86 (m, 1H), 4.31-4.30 (m, 1H), 4.56-4.32 (d, J=1.6 Hz, 2H),5.05 (s, 2H), 5.62 (bs, 1H), 7.35-7.29 (m, 5H), 7.88 (s, 1H), 8.04 (s,1H). The title compound was shown to inhibit hT2R08 bitter receptor andhad an IC₅₀ of 0.76 μM.

Example 10-482-(1-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-2,5-dioxoimidazolidin-4-yl)-N-phenylacetamide

3-(1-(1-methylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-2,5-dioxoimidazolidin-4-yl)aceticacid (example 10-44) (100 mg, 0.3 mmol), aniline (33 mg, 0.36 mmol),Pybop (187 mg, 0.36 mmol) and triethyl amine (0.05 mL, 0.36 mmol) weremixed in DMF (1 mL). The reaction stirred at 65° C. for 4 hours. Thereaction was allowed to cool to room temperature and then diluted withethyl acetate (2 mL). The organic phase was washed with saturated sodiumbicarbonate solution (2×, 2 mL) and then with saturated NaCl solution (1ml). The organic phase was extracted, dried over anhydrous Na₂SO₄, andfiltered. The crude product was re-suspended in MeOH (1 mL) and purifiedby reversed phase HPLC (5-95% acetonitrile in H₂O; 16 minute gradient).The pure fractions were combined and solvent was removed on the rotovapto afford2-(1-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-2,5-dioxoimidazolidin-4-yl)-N-phenylacetamideas a white solid (50%). ¹H NMR (CDCl₃, 400 MHz): δ 2.18 (s, 3H), 2.40(s, 3H), 2.72 (m, 1H), 3.14-3.13 (d, 1H, J=4 Hz), 4.54-4.51 (d, J=8 Hz1H), 5.04 (s, 2H), 6.53 (bs, 1H), 7.15-7.13 (m, 1H), 7.33-7.22 (m, 2H),7.47-7.45 (d, J=8 Hz, 2H), 7.78 (bs, 1H), 7.09 (s, 1H), 8.05 (s, 1H).The title compound was shown to inhibit hT2R08 bitter receptor and hadan IC₅₀ of 0.75 μM.

Example 10-49N-benzyl-2-(1-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-2,5-dioxoimidazolidin-4-yl)acetamide

Prepared as in Example 10-48 from3-(1-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-2,5-dioxoimidazolidin-4-yl)aceticacid (example 10-44) and benzyl amine. Yield: 30%. ¹H NMR (CDCl₃, 400MHz): δ 2.18 (s, 3H), 2.41 (s, 3H), 2.56-2.52 (m, 1H, J=16 Hz),2.56-2.52 (m, 1H), 3.00-2.96 (m, 1H), 4.45-4.44 (d, J=5.6 Hz, 2H), 5.04(s, 2H), 5.96 (bs, 1H), 6.36 (bs, 1H), 7.36-7.25 (m, 5H), 7.90 (s, 1H,8.05 (s, 1H). The title compound was shown to inhibit hT2R08 bitterreceptor and had an IC₅₀ of 1.3 μM.

Example 10-502-(1-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-2,5-dioxoimidazolidin-4-yl)-N-(3-methoxybenzyl)acetamide

Prepared as in Example 10-48 from3-(1-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-2,5-dioxoimidazolidin-4-yl)aceticacid (example 10-44) and (3-methoxyphenyl)methanamine. Yield: 50%.LC/MS; expected 453; found 453.1 The title compound was shown to inhibithT2R08 bitter receptor and had an IC₅₀ of 1.7 μM.

Example 10-513-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-1-methylimidazolidine-2,4-dione

3-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)imidazolidine-2,4-dione(example 10-1) (50 mg, 0.182 mmol) and cesium carbonate (60 mg, 0.185mmol) were mixed in DMF (1 mL) for 15 minutes under a nitrogenatmosphere at room temperature. Then, iodomethane (14 mg, 0.185 mmol)was added and the reaction continued to stir for an addition 2 hours.H₂O (2 mL) was added and the product was extracted with ethyl acetate (1mL, 2×). The organic phase was collected and washed with saturatedsodium bicarbonate solution (2 ml, 2×), dried, and filtered. Solvent wasremoved under a stream of nitrogen and then further dried under highvacuum to afford3-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-1-methylimidazolidine-2,4-dioneas a white solid (42 mg, 80%). Yield: 80%. ¹H NMR (CDCl₃, 400 MHz): δ2.18 (s, 3H), 2.41 (s, 3H), 3.06 (s, 3H), 3.95 (s, 2H), 5.05 (s, 2H),7.89 (s, 1H), 8.05 (s, 1H). The title compound was shown to inhibithT2R08 bitter receptor and had an IC₅₀ of 0.58 μM.

Example 10-523-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-1,5,5-trimethylimidazolidine-2,4-dione

3-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-5-methylimidazolidine-2,4-dione(example 10-41) (50 mg, 0.173 mmol) and 60% NaH (8 mg, 0.190 mmol) weremixed in DMF (1 mL) for 30 minutes. MeI (0.04 mL, 0.190 mmol) was addedand the reaction was stirred an additional 4 hours. The reaction wasacidified with 1N HCl and diluted with ethyl acetate (2 mL). The organicphase was dried, filtered and solvent was removed under a stream ofnitrogen. The crude product was re-suspended in MeOH (1 mL) and purifiedby reversed phase HPLC (5 to 95% acetonitrile in H₂O: 16 minutesgradient). The pure fractions were combined and solvent removed undervacuum to afford3-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-1,5,5-trimethylimidazolidine-2,4-dioneas a white solid (25 mg, 50%). NMR (CDCl₃, (CDCl₃, 400 MHz): δ 1.45 (s,6H), 2.19 (s, 3H), 2.42 (s, 3H), 2.94 (s, 3H), 5.05 (s, 2H), 7.92 (s,1H), 8.08 (s, 1H). The title compound was shown to inhibit hT2R08 bitterreceptor and had an IC₅₀ of 0.80 μM.

Example 10-531-benzyl-3-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)imidazolidine-2,4-dione

Prepared as in example 10-5 from3-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)imidazolidine-2,4-dione(example 10-1) and benzyl bromide. Yield: 40%. ¹H NMR (CDCl₃, 400 MHz):δ 2.18 (s, 3H), 2.42 (s, 3H), 3.84 (s, 2H), 4.61 (s, 2H), 5.06 (s, 2H),7.40-7.27 (m, 5H), 7.92 (s, 1H), 8.08 (s, 1H). The title compound wasshown to inhibit hT2R08 bitter receptor and had an IC₅₀ of 0.09 μM.

Example 10-543-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-1-(pyridin-2-ylmethyl)imidazolidine-2,4-dione

Prepared as in Example 10-5 from3-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)imidazolidine-2,4-dioneand 2-(bromomethyl)pyridine. Yield: 50%. ¹H NMR (CDCl₃, 400 MHz): δ 2.19(s, 3H), 2.41 (s, 3H), 4.12 (s, 2H), 4.71 (s, 2H), 5.05 (s, 2H),7.72-7.23 (m, 4H), 7.92 (s, 1H) 8.08 (s, 1H). The title compound wasshown to inhibit hT2R08 bitter receptor and had an IC₅₀ of 0.68 μM.

Example 10-551-((3,5-dimethylisoxazol-4-yl)methyl)-3-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)imidazolidine-2,4-dione

Prepared as in Example 10-5 from3-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)imidazolidine-2,4-dioneand 4-(chloromethyl)-3,5-dimethylisoxazole. Yield: 50%. ¹NMR (CDCl₃, 400MHz): 2.19 (s, 3H), 2.27 (s, 3H), 2.43-2.42 (d, J=5.2 Hz, 6H), 3.82 (s,2H), 4.40 (s, 2H), 5.05 (s, 2H), 7.92 (s, 1H), 8.05 (s, 1H). The titlecompound was shown to inhibit hT2R08 bitter receptor and had an IC₅₀ of0.04 μM.

Example 10-563-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-5-(3-methoxybenzyl)imidazolidine-2,4-dione

1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-amine hydrochloride(400 mg, 2.08 mmol), dipyridin-2-yl carbonate (450 mg, 2.08 mmol) andtriethylamine (0.290 mL, 2.08 mmol) were stirred in dichloromethane (7mL) for 12 hours at room temperature. The reaction was concentratedunder vacuum to afford4-((4-isocyanato-1H-pyrazol-1-yl)methyl)-3,5-dimethylisoxazole as anoff-white solid in quantitative yield. Ethanol (1 mL) was added alongwith methyl 2-amino-3-(3-methoxyphenyl)propanoate (68 mg, 0.327 mmol)and triethylamine (0.064 mL, 0.461 mmol). The reaction was stirred atreflux for 12 hours, then allowed to cool to room temperature. Thesolvent was removed under a stream of nitrogen. The crude product wasre-suspended in MeOH (1 mL) and purified by reversed phase HPLC (5 to95% acetonitrile in H₂O: 16 minutes gradient). The pure fractions werecombined and solvent removed under vacuum to afford3-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-5-(3-methoxybenzyl)imidazolidine-2,4-dioneas a white solid yield: 50% ¹H NMR (CDCl₃, 400 MHz): δ 2.19 (s, 3H),2.41 (s, 3H), 3.34-3.33 (d, J=3.2 Hz, 1H), 3.31-3.29 (d, J=8 Hz, 1H),3.76 (s, 3H), 4.33-4.30 (m, 1H), 5.05 (s, 2H), 5.95 (bs, 1H), 7.25-7.21(t, 1H), 6.82-6.78 (m, 3H), 7.85 (s, 1H), 7.99 (s, 1H). The titlecompound was shown to inhibit hT2R08 bitter receptor and had an IC₅₀ of0.13 μM.

Example 10-575-(cyclohexylmethyl)-3-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)imidazolidine-2,4-dione

Prepared as in Example 10-56 from1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-amine hydrochloride(example 10-41a) and methyl 2-amino-3-cyclohexylpropanoate. Yield: 30%.¹H NMR (CDCl₃, 400 MHz): δ 1.06-0.95 (m, 2H), 1.29-1.15 (m, 3H),1.60-1.50 (1H) 1.77-1.67 (7H), 1.91-1.85 (m, 1H), 2.19 (s, 3H), 2.41 (s,3H), 4.19-4.15 (m, 1H), 5.05 (s, 2H), 6.01 (bs, 1H), 7.91 (s, 1H), 8.05(s, 1H). The title compound was shown to inhibit hT2R08 bitter receptorand had an IC₅₀ of 0.96 μM.

Example 10-585-(cyclopentylmethyl)-3-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)imidazolidine-2,4-dione

Prepared as in Example 10-56 from1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-amine hydrochloride(example 10-41a) and methyl 2-amino-3-cyclopentylpropanoate. Yield: 50%.¹H NMR (CDCl₃, 400 MHz): δ 1.20-1.14 (m, 3H), 1.68-1.55 (m, 6H),2.04-1.92 (m, 2H), 2.19 (s, 3H), 2.42 (s, 3H), 4.14-4.11 (m, 1H), 5.05(s, 2H), 5.52 (bs, 1H), 7.90 (s, 1H), 8.06 (s, 1H). The title compoundwas shown to inhibit hT2R08 bitter receptor and had an IC₅₀ of 0.31 μM.

Example 10-593-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-5-(4-hydroxybenzyl)imidazolidine-2,4-dione

Prepared as in Example 10-56 from1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-amine hydrochloride(example 10-41a) and methyl 2-amino-3-(4-hydroxyphenyl)propanoate.Yield: 50%. ¹H NMR (CDCl₃, 400 MHz): δ 2.41 (s, 3H), 2.85 (s, 3H),3.26-3.25 (d, 1H, J=4 Hz), 3.23-3.22 (d, J=4 Hz, 1H) 4.31-4.28 (m, 1H),5.04 (s, 2H), 5.77-5.74 (bs, 1H), 7.07-7.04 (d, J=12 Hz, 2H), 6.75-6.73(d, J=8 Hz, 2H), 7.06 (s, 1H), 7.97 (s, 1H), 9.43 (bs, 1H). The titlecompound was shown to inhibit hT2R08 bitter receptor and had an IC₅₀ of0.33 μM.

Example 10-605-(3,4-dihydroxybenzyl)-3-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)imidazolidine-2,4-dione

Prepared as in Example 10-56 from1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-amine hydrochloride(example 10-41a) and methyl 2-amino-3-(3,4-dihydroxyphenyl)propanoate.Yield: 50%. MS M+H calculated 398.1; found 398.1. The title compound wasshown to inhibit hT2R08 bitter receptor and had an IC₅₀ of 0.51 μM.

Example 10-613-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-5-isobutylimidazolidine-2,4-dione

Prepared as in Example 10-41 from1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-amine hydrochloride(example 10-41a) and ethyl 2-isocyanato-4-methylpentanoate. Yield: 50%.¹H NMR (CDCl₃, 400 MHz): δ 1.01-0.98 (m, 8H), 1.87-1.82 (m, 1H), 2.19(s, 3H), 2.41 (s, 3H), 4.13-4.12 (t, 1H), (5.05 (s, 2H), 5.70 (bs, 1H),7.90 (s, 1H), 8.05 (s, 1H). The title compound was shown to inhibithT2R08 bitter receptor and had an IC₅₀ of 1.0 μM.

Example 10-623-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-5-isopropylimidazolidine-2,4-dione

Prepared as in Example 10-41 from1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-amine hydrochloride(example 10-41a) and ethyl 2-isocyanato-3-methylbutanoate. Yield: 30%.¹H NMR (CDCl₃, 400 MHz): δ 0.96-0.94 (d, 3H, J=7.2 Hz), 1.09-1.07 (d,3H, J=8 Hz), 2.19 (s, 3H), 2.26-2.22 (m, 1H), 2.40 (s, 3H), 4.02 (s,1H), 5.05 (s, 2H), 5.53 (bs, 1H), 7.90 (s, 1H), 8.05 (s, 1H). The titlecompound was shown to inhibit hT2R08 bitter receptor and had an IC₅₀ of1.1 μM.

Example 10-63(Z)-5-benzylidene-3-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)imidazolidine-2,4-dione

3-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)imidazolidine-2,4-dione(example 10-1) (275 mg, 1 mmol), benzaldehyde (140 mg, 1.3 mmol), andsodium acetate (205 mg, 2.5 mmol), in glacial acetic acid (3 mL) wereirradiated in the microwave reactor for 7 hours at 185° C. Upon coolingthe mixture was diluted with H₂O (100 mL) and extracted with ethylacetate (3×, 50 mL). The combined organic extracts were washed withsaturated aqueous sodium carbonate solution, dried over sodium sulfate,filtered and concentrated. the solid product was triturated with ethylacetate/hexanes (1/1) and dried under high vacuum to afford(Z)-5-benzylidene-3-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)imidazolidine-2,4-dione(173 mg, 48%) as a light yellow solid. ¹H NMR (DMSO-d₆, 400 MHz): δ 2.14(s, 3H), 2.40 (s, 3H), 6.59 (s, 1H), 5.20 (s, 2H), 7.33-7.40 (m, 3H),7.66 (s, 2H), 7.81 (s, 1H), 8.21 (s, 1H), 11.01 (bs, 1H). The titlecompound was shown to inhibit hT2R08 bitter receptor and had an IC₅₀ of0.34 μM.

Example 10-644-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-1-methyl-1,2,4-triazolidine-3,5-dione

Ethyl4-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-2-methyl-3,5-dioxo-1,2,4-triazolidine-1-carboxylate(3.2 g, 8.8 mmol) was stirred in a (1/1) mixture of MeOH/1N aqueous NaOH(100 mL) at ambient temperature for 30 minutes. The mixture wasacidified with aqueous 1N HCl (150 mL), extracted with ethyl acetate(3×, 100 mL), dried over sodium sulfate, filtered and concentrated toafford4-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-1-methyl-1,2,4-triazolidine-3,5-dione(2.3 g, 89%) as a yellow solid. ¹H NMR (DMSO-d₆, 400 MHz): δ2.18 (s,3H), 2.44 (s, 3H), 3.05 (s, 3H), 5.17 (s, 2H), 7.94 (s, 1H), 8.13 (s,1H).

Example 10-64a Ethyl4-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-2-methyl-3,5-dioxo-1,2,4-triazolidine-1-carboxylate

Ethyl chloroformate (1.3 g, 12 mmol) was added to a mixture ofN-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-1-methylhydrazinecarboxamide(example 10-64b) (2.5 g, 9 mmol) and triethylamine (1.2 g, 12 mmol) inacetonitrile (100 ml). The mixture was refluxed for 1 hour, cooled thendiluted with 1N aqueous HCl (150 mL) and extracted with ethyl acetate(3×, 75 mL). The combined organic extracts were dried over sodiumsulfate, filtered and concentrated on the rotovap. The solid wastriturated with ethyl acetate/hexanes (1/3) and dried under high vacuumto afford ethyl4-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-2-methyl-3,5-dioxo-1,2,4-triazolidine-1-carboxylate(3.2 g, 94%) as a white solid. ¹H NMR (DMSO-d₆, 400 MHz): δ1.28 (t,J=7.2 Hz, 3H), 2.13 (s, 3H), 2.40 (s, 3H), 3.24 (s, 3H), 4.30 (t, J=7.2Hz, 2H), 5.21 (s, 2H), 7.73 (m, 1H), 8.16 (s, 1H).

Example 10-64bN-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-1-methylhydrazinecarboxamide

1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazole-4-carbonyl azide(example 10-1a) (1.25 g, 5.3 mmol) was stirred in toluene (30 mL) atreflux temperature for 40 minutes. The mixture was cooled to ambienttemperature and methyl hydrazine (0.3 mL, 260 mg, 5.6 mmol) was addedand the mixture was refluxed for 30 minutes. After cooling the reactionto room temperature the solvent was removed on the rotovap and the solidproduct was triturated with ethyl acetate/hexanes (2/5) and dried underhigh vacuum to affordN-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-1-methylhydrazinecarboxamide(1.1 g, 79%) as a white solid. ¹H NMR (DMSO-d₆, 400 MHz): δ2.09 (s, 3H),2.36 (s, 3H), 2.98 (s, 3H), 4.61 (s, 2H), 5.02 (s, 2H), 7.42 (s, 1H),7.72 (m, 1H), 8.78 (s, 1H).

Example 10-651-Benzyl-4-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-2-methyl-1,2,4-triazolidine-3,5-dione

4-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-1-methyl-1,2,4-triazolidine-3,5-dione(example 10-64) (785 mg, 2.7 mmol) was dissolved in acetonitrile (50mL). Triethylamine (1 g, 10 mmol) and benzyl bromide (510 mg, 3 mmol)were added and the reaction was stirred at ambient temperature for 12hours. The mixture was then concentrated on the rotovap, dissolved inmethanol (5 mL) and purified by reversed phase HPLC (5-95% acetonitrilein H₂O: 25 minute gradient). The pure fractions were pooled andconcentrated and the product was recrystallized form ethanol to afford1-benzyl-4-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-2-methyl-1,2,4-triazolidine-3,5-dione(210 mg, 20%) as a white solid. ¹H NMR (DMSO-d₆, 400 MHz): δ2.13 (s,3H), 2.39 (s, 3H), 3.09 (s, 3H), 4.81 (s, 2H), 5.18 (s, 2H), 7.30-7.35(m, 5H), 7.76 (s, 1H), 8.18 (s, 1H). MS M+H calculated 381.1; found381.1. Melting point: 124-126° C. The title compound was shown toinhibit hT2R08 bitter receptor and had an IC₅₀ of 0.02 μM.

Example 10-661-Benzyl-4-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-2-methyl-1,2,4-triazolidine-3,5-dione

Prepared as in Example 10-65 from4-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-1-methyl-1,2,4-triazolidine-3,5-dione(example 10-64) and 1-(2-bromoethyl)-4-fluorobenzene Yield: 14%. ¹H NMR(CDCl₃, 400 MHz): δ2.18 (s, 3H), 2.40 (s, 3H), 2.87 (t, J=6.8 Hz, 2H),3.14 (s, 3H), 3.83 (t, J=7.2 Hz, 2H), 5.03 (s, 2H), 6.95 (t, J=8.4 Hz,2H), 7.14 (t, J=8 Hz, 2H), 7.77 (s, 1H), 7.95 (s, 1H). The titlecompound was shown to inhibit hT2R08 bitter receptor and had an IC₅₀ of0.01 μM.

Example 10-674-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-1-methyl-2-(2-phenoxyethyl)-1,2,4-triazolidine-3,5-dione

Prepared as in Example 10-65 from4-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-1-methyl-1,2,4-triazolidine-3,5-dione(example 10-64) and (2-bromoethoxy)benzene. Yield: 20%. ¹H NMR (DMSO-d₆,400 MHz): δ2.13 (s, 3H), 2.40 (s, 3H), 3.15 (s, 3H), 3.99 (t, J=4.4 Hz,2H), 4.13 (t, J=4.8 Hz, 2H), 5.20 (s, 2H), 6.80 (d, J=8 Hz, 2H), 6.90(t, J=7.1 Hz, 1H), 7.22 (t, J=8 Hz, 2H), 7.75 (s, 1H), 8.17 (s, 1H). Thetitle compound was shown to inhibit hT2R08 bitter receptor and had anIC₅₀ of 0.031 μM.

Example 10-684-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-1,2,4-triazolidine-3,5-dione

1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazole-4-carbonyl azide(example 10-1a) (1 g, 4.1 mmol) was refluxed in toluene (100 mL) for onehour. The reaction was cooled to room temperature and ethylhydrazinecarboxylate (0.45 g, 43 mmol) was added. The reaction washeated to reflux and stirred for 1 hour, then cooled and concentrated onthe rotovap. The residue was taken up in ethanol (100 ml) and potassiumcarbonate (100 mg) was added. The mixture was refluxed for 12 hours,then filtered, cooled to ambient temperature, and neutralized withacetic acid (ca. 7 drops). The solvent was removed on the rotovap andthe resulting solid was triturated with ethyl acetate/hexanes (1/9) toafford4-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-1,2,4-triazolidine-3,5-dione(0.98 g, 85%) as an off white solid. ¹H NMR (CDCl₃, 400 MHz): δ2.18 (s,3H), 2.41 (s, 3H), 4.98 (s, 2H) 7.16 (s, 1H), 7.38 (s, 1H).

Example 10-694-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-1,2-dimethyl-1,2,4-triazolidine-3,5-dione

4-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-1,2,4-triazolidine-3,5-dione(example 10-68) (100 mg, 0.36 mmol), methyl iodide (141 mg, 1 mmol), andcesium carbonate (325 mg, 1 mmol) were stirred in a 2/1 mixture ofacetonitrile/DMF (5 mL) at ambient temperature for 2 hours. The mixturewas diluted with aqueous 1N HCl (100 mL) and extracted with ethylacetate (3×, 50 mL). The combined organic extracts were dried oversodium sulfate, concentrated and the crude residue taken up in MeOH andpurified by reversed phase HPLC (5 to 95% acetonitrile in H₂O: 25 minutegradient). The pure fractions were pooled and concentrated to afford4-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-1,2-dimethyl-1,2,4-triazolidine-3,5-dione(89 mg, 80%) as a clear semi-solid. NMR (CDCl₃, 400 MHz): δ2.18 (s, 3H),2.41 (s, 3H), 3.22 (s, 6H), 5.04 (s, 2H), 7.88 (s, 1H), 8.03 (s, 1H).The title compound was shown to inhibit hT2R08 bitter receptor and hadan IC₅₀ of 0.6 μM.

Example 10-701,2-dibenzyl-4-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-1,2,4-triazolidine-3,5-dione

Prepared as in Example 10-65 from4-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-1,2-dimethyl-1,2,4-triazolidine-3,5-dione(example 10-69) and benzyl bromide. Yield: 69%. ¹H NMR (CDCl₃, 400 MHz):δ2.14 (s, 3H), 2.36 (s, 3H), 4.65 (s, 4H), 4.99 (s, 2H), 7.06-7.08 (m,4H), 7.19-7.25 (m, 6H), 7.86 (s, 1H), 8.02 (s, 1H). The title compoundwas shown to inhibit hT2R08 bitter receptor and had an IC₅₀ of 0.8 μM.

Example 10-713-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-1-((6-(hydroxymethyl)pyridin-2-yl)methyl)imidazolidine-2,4-dione

Prepared as in example 10-5 from3-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)imidazolidine-2,4-dioneand 6-bromomethyl-2-pyridinemethanol. Yield: 35%. MS M+H calculated397.2; found 397.2. The title compound was shown to inhibit hT2R08bitter receptor and had an IC₅₀ of 0.72 μM.

Example 10-721-((3,4-dimethoxypyridin-2-yl)methyl)-3-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)imidazolidine-2,4-dione

Prepared as in example 10-5 from3-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)imidazolidine-2,4-dioneand 3,4-dimethoxy-2-chloromethyl pyridine hydrochloride. Yield: 26%. MSM+H calculated 360.2; found 360.2. The title compound was shown toinhibit hT2R08 bitter receptor and had an IC₅₀ of 1.0 μM.

Example 10-733-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-1-((6-(tetrahydrofuran-2-yl)methyl)imidazolidine-2,4-dione

Prepared as in example 10-52 from3-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)imidazolidine-2,4-dioneand tetrahydrofurfuryl bromide. Yield: 28%. MS M+H calculated 427.2;found 427.2. The title compound was shown to inhibit hT2R08 bitterreceptor and had an IC₅₀ of 1.4 μM.

Example 10-741-(cyclohexylmethyl)-3-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)imidazolidine-2,4-dione

Prepared as in example 10-52 from3-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)imidazolidine-2,4-dione(example 10-1) and bromomethylcyclohexane. Yield: 20%. ¹H NMR (DMSO-d₆,400 MHz): δ0.88 (q, J=10.4 Hz, 2H), 1.09-1.19 (m, 3H), 1.58-1.65 (m,6H), 2.12 (s, 3H), 2.38 (s, 3H), 3.13 (d, J=7.2 Hz, 2H), 4.06 (s, 2H),5.17 (s, 2H), 7.75 (s, 1H), 8.14 (s, 1H). MS M+H calculated 372.2; found372.1. The title compound was shown to inhibit hT2R08 bitter receptorand had an IC₅₀ of 0.28 μM.

Example 10-753-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-1-((6-methoxypyridin-2-yl)methyl)imidazolidine-2,4-dione

3-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)imidazolidine-2,4-dione(100 mg, 0.4 mmol), (6-methoxy-pyridin-2-yl)-methanol (example 10-75a)(101 mg, 0.7 mmol), tributylphosphine (147 mg, 0.7 mmol), and1,1″″-azobis(N,N-dimethylformamide) (125 mg, 0.7 mmol) were dissolved inTHF (5 mL) and stirred at room temperature for 15 hours. The reactionwas diluted with brine (100 mL) and extracted with ethyl acetate (2×,100 mL). The combined organic extracts were dried over magnesiumsulfate, filtered and concentrated on the rotovap. The residue was takenup in methanol (5 mL) and purified by reversed phase HPLC (5 to 95%acetonitrile in H₂O: 25 minute gradient). The pure fractions werecombined, concentrated then re-dissolved in ethyl acetate/hexane (1:9).The solution was cooled at 5° C. for 15 hrs, where a white solid formed.The precipitate was collected to afford3-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-1-((6-methoxypyridin-2-yl)methyl)imidazolidine-2,4-dione(5 mg, 4%) as a white solid. ¹H NMR (DMSO-d₆, 400 MHz): δ2.11 (s, 3H),2.37 (s, 3H), 3.74 (s, 3H), 4.17 (s, 2H), 4.54 (s, 2H), 5.17 (s, 2H),6.69 (d, J=7.6 Hz, 1H), 6.96 (d, J=6.8 Hz, 1H), 7.67 (d, J=6.8 Hz, 1H),7.76 (s, 1H), 8.17 (s, 1H). MS M+H calculated 397.2; found 397.2. Thetitle compound was shown to inhibit hT2R08 bitter receptor and had anIC₅₀ of 0.23 μM.

Example 10-75a (6-methoxypyridin-2-yl)methanol

Methyl-6-methoxypyridine-2-carboxylate (2 g, 11.96 mmol) in anhydrousmethanol (20 mL) was cooled to 0° C. under nitrogen and sodiumborohydride (1.36 g, 35.89 mmol) was slowly added to the solution. Thereaction was left stirring at 0° C. for 30 minutes, then allowed to warmup to room temperature for 1 hour. The reaction was quenched with waterand concentrated on the rotovap. The reaction was diluted with brine(100 mL) and extracted with dichloromethane/2-propanol solution (2:1)(3×, 150 mL). The combined organic extracts were dried over magnesiumsulfate, filtered and concentrated on the rotovap to afford(6-methoxypyridin-2-yl)methanol (500 mg, 30%) as an oil. MS M+Hcalculated 140.1; found 140.1.

Example 10-763-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-1-pyrazol-4-yl)-1-(2-methoxyphenethyl)imidazolidine-2,4-dione

Prepared as in example 10-52 from3-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)imidazolidine-2,4-dione(example 6) and 2-methoxyphenethyl bromide. Yield: 52%. ¹H NMR (DMSO-d₆,400 MHz): δ2.11 (s, 3H), 2.38 (s, 3H), 2.80 (t, J=7.2 Hz, 2H), 3.51 (t,J=7.2 Hz, 2H), 3.75 (s, 3H), 4.03 (s, 2H), 5.17 (s, 2H), 6.85 (t, J=7.2Hz, 1H), 6.95 (d, J=8.4 Hz, 1H), 7.16-7.21 (m, 2H), 7.73 (s, 1H), 8.13(s, 1H). MS M+H calculated 410.2; found 410.1. Melting point: 97-98° C.The title compound was shown to inhibit hT2R08 bitter receptor and hadan IC₅₀ of 0.14 μM.

Example 10-773-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-1-(3-fluorophenethyl)imidazolidine-2,4-dione

Prepared as in example 10-52 from3-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)imidazolidine-2,4-dione(example 10-1) and 3-fluorophenethyl bromide. Yield: 22%. ¹H NMR(DMSO-d₆, 400 MHz):

2.11 (s, 3H), 2.38 (s, 3H), 2.80 (t, J=7.2 Hz, 2H), 3.57 (t, J=7.2 Hz,2H), 4.06 (s, 2H), 5.17 (s, 2H), 6.85 (dt, J=8.4, 2.0 Hz, 1H), 7.11 (t,J=8.4 Hz, 2H), 7.32 (q, J=7.8 Hz, 1H), 7.73 (s, 1H), 8.12 (s, 1H). MSM+H calculated 398.2; found 398.1. Melting point: 110-111° C. The titlecompound was shown to inhibit hT2R08 bitter receptor and had an IC₅₀ of0.06 μM.

Example 10-783-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-1-phenethylimidazolidine-2,4-dione

Prepared as in example 10-52 from3-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)imidazolidine-2,4-dione(example 10-1) and phenethylbromide. Yield: 37%. ¹H NMR (DMSO-d₆, 400MHz): δ2.11 (s, 3H), 2.38 (s, 3H), 2.83 (t, J=6.4 Hz, 2H), 3.55 (t,J=7.4 Hz, 2H), 4.03 (s, 2H), 5.17 (s, 2H), 7.18-7.30 (m, 5H), 7.73 (s,1H), 8.13 (s, 1H). MS M+H calculated 380.2; found 380.1. Melting point:95-96° C. The title compound was shown to inhibit hT2R08 bitter receptorand had an IC₅₀ of 0.14 μM.

Example 10-793-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-1-(4-methoxyphenethyl)imidazolidine-2,4-dione

Prepared as in example 10-52 from3-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)imidazolidine-2,4-dione(example 10-1) and 4-methoxyphenethyl bromide. Yield: 32%. ¹H NMR(DMSO-d₆, 400 MHz): δ2.11 (s, 3H), 2.38 (s, 3H), 2.78 (t, J=7.4 Hz, 2H),3.50 (t, J=7.4 Hz, 2H), 3.69 (s, 3H), 4.02 (s, 2H), 5.16 (s, 2H), 6.84(d, J=8.4 Hz, 2H), 7.15 (d, J=8.4 Hz, 2H), 7.73 (s, 1H), 8.12 (s, 1H).MS M+H calculated 410.18; found 410.2. The title compound was shown toinhibit hT2R08 bitter receptor and had an IC₅₀ of 0.04 μM.

Example 10-803-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-1-(2-naphthalen-1-yl)ethyl)imidazolidine-2,4-dione

Prepared as in example 10-52 from3-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)imidazolidine-2,4-dione(example 10-1) and 1-(2-bromoethyl)naphthalene. Yield: 20%. MS M+Hcalculated 430.18; found 430.2. The title compound was shown to inhibithT2R08 bitter receptor and had an IC₅₀ of 1.27 μM.

Example 10-811-(2-chlorophenethyl)-3-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)imidazolidine-2,4-dione

Prepared as in example 10-52 from3-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)imidazolidine-2,4-dione(example 10-1) and 2-chlorophenethyl bromide. Yield: 25%. MS M+Hcalculated 414.13; found 414.2. The title compound was shown to inhibithT2R08 bitter receptor and had an IC₅₀ of 0.25 μM.

Example 10-821-(3-chlorophenethyl)-3-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)imidazolidine-2,4-dione

Prepared as in example 10-52 from3-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)imidazolidine-2,4-dione(example 10-1) and 3-chlorophenethyl bromide. Yield: 27%. MS M+Hcalculated 414.13; found 414.2. The title compound was shown to inhibithT2R08 bitter receptor and had an IC₅₀ of 0.20 μM.

Example 10-833-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-1-(2-fluorophenethyl)imidazolidine-2,4-dione

Prepared as in example 10-52 from3-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)imidazolidine-2,4-dione(example 10-1) and 2-fluorophenethyl bromide. Yield: 24%. MS M+Hcalculated 398.16; found 398.1. The title compound was shown to inhibithT2R08 bitter receptor and had an IC₅₀ of 0.13 μM.

Example 10-843-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-1-(4-fluorophenethyl)imidazolidine-2,4-dione

Prepared as in example 10-52 from3-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)imidazolidine-2,4-dione(example 10-1) and 4-fluorophenethyl bromide. Yield: 34%. MS M+Hcalculated 398.16; found 398.1. The title compound was shown to inhibithT2R08 bitter receptor and had an IC₅₀ of 0.01 μM.

Example 10-853-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-1-(3-methoxyphenethyl)imidazolidine-2,4-dione

Prepared as in example 10-52 from3-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)imidazolidine-2,4-dione(example 10-1) and 3-methoxyphenethyl bromide. Yield: 34%. MS M+Hcalculated 410.18; found 410.1. The title compound was shown to inhibithT2R08 bitter receptor and had an IC₅₀ of 0.16 μM.

Example 10-863-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-1-(4-hydroxyphenethyl)imidazolidine-2,4-dione

Prepared as in example 10-52 from3-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)imidazolidine-2,4-dione(example 10-1) and 4-hydroxyphenethyl bromide. Yield: 31%. MS M+Hcalculated 396.16; found 396.1. The title compound was shown to inhibithT2R08 bitter receptor and had an IC₅₀ of 0.41 μM.

Example 10-871-(3,4-dimethoxyphenethyl)-3-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)imidazolidine-2,4-dione

Prepared as in example 10-52 from3-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)imidazolidine-2,4-dione(example 10-1) and 3,4-dimethoxyphenethyl bromide. Yield: 36%. MS M+Hcalculated 440.19; found 440.2. The title compound was shown to inhibithT2R08 bitter receptor and had an IC₅₀ of 0.26 μM.

Example 10-881-benzyl-3-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)imidazolidin-2-one

1-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)imidazolidin-2-one(example 10-88a) (50 mg, 0.19 mmol) and 60% sodium hydride (8 mg, 0.21mmol) in DMF (3 mL) were stirred at room temperature for 15 minutes thencooled to 0° C. Benzyl bromide (33 mg, 0.19 mmol) was added to themixture and allowed to warm up at room temperature for 2 hours. Thereaction was quenched with methanol and concentrated on the rotovap. Thereaction was diluted with brine (50 mL) and extracted withdichloromethane (2×, 50 mL). The combined organic extracts were driedover magnesium sulfate, filtered and concentrated on the rotovap. Theresidue was purified by silica column chromotography (100% to 90%dichloromethane in methanol: 30 minute gradient) to afford1-benzyl-3-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)imidazolidin-2-one(21 mg, 31%) as a white solid. MS M+H calculated 352.17; found 352.2.The title compound was shown to inhibit hT2R08 bitter receptor and hadan IC₅₀ of 0.71 μM.

Example 10-88a1-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)imidazolidin-2-one

1-(2-chloroethyl)-3-(1-((3,5-dimethylisoxazol-4-yl)methyl)1H-pyrazol-4-yl)urea(example 10-88b) (115 mg, 0.39 mmol) and 60% sodium hydride (17 mg, 0.42mmol) in DMF (2 mL) were stirred at 0° C. for 15 minutes then allowed towarm up to room temperature with stirring for 2 hours. The reaction wasquenched with methanol and concentrated on the rotovap. The reaction wasdiluted with brine (50 mL) and extracted with dichloromethane (2×, 50mL). The combined organic extracts were dried over magnesium sulfate,filtered and concentrated on the rotovap. The residue was purified bysilica column chromotography (100% to 90% dichloromethane in methanol:30 minute gradient) to afford1-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)imidazolidin-2-one(98 mg, 97%) as a white solid. ¹H NMR (DMSO-d₆, 400 MHz): δ2.10 (s, 3H),2.37 (s, 3H), 3.35-3.39 (m, 2H), 3.61 (d, J=8.8 Hz, 1H), 3.63 (d, J=10.4Hz, 1H), 5.07 (s, 2H), 6.71 (s, 1H), 7.42 (s, 1H), 7.74 (s, 1H). MS M+Hcalculated 262.12; found 262.1.

Example 10-88b1-(2-chloroethyl)-3-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)urea

1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-amine (419 mg, 2.18mmol) and 2-chloroethyl isocyanate (230 mg, 2.18 mmol) in acetonitrile(5 mL) were heated at 65° C. for 16 hours. The reaction was cooled toroom temperature and concentrated on the rotovap. The residue waspurified by silica column chromotography (100% to 90% dichloromethane inmethanol: 30 minute gradient), dried and triturated with ethylacetate/hexanes (1/9) to afford(1-(2-chloroethyl)-3-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)urea(258 mg, 40%) as a yellow solid. MS M+H calculated 298.10; found 298.1

Example 10-891-benzyl-4-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-2-(methoxymethyl)-1,2,4-triazolidine-3,5-dione

Prepared as in example 10-91 from1-benzyl-4-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-1,2,4-triazolidine-3,5-dione(example 10-91a) and bromomethyl methyl ether. Yield: 18%. MS M+Hcalculated 411.17; found 411.2. The title compound was shown to inhibithT2R08 bitter receptor and had an IC₅₀ of 0.02 μM.

Example 10-901-((1,3-dimethyl-1H-pyrazol-4-yl)methyl)-3-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)imidazolidine-2,4-dione

3-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)imidazolidine-2,4-dione(10a)(200 mg, 0.7 mmol), 4-(chloromethyl)-1,3-dimethyl-1H-pyrazole (144mg, 1 mmol), and cesium carbonate (325 mg, 1 mmol) were dissolved in 2mL DMF and irradiated in the microwave reactor at 165° C. for 5 minutes.The reaction was cooled to room temperature, and salt precipitate wasremoved by filtration. The clear solution containing crude product wasobtained and was purified by HPLC (10 to 95% Acetonitrile/water; 25minutes) to afford1-((1,3-dimethyl-1H-pyrazol-5-yl)methyl)-3-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)imidazolidine-2,4-dione(150 mg, 53%) as a light brown solid. ¹2.09H NMR (DMSO, 400 MHz): δ (s,3H), 2.14 (s, 3H), 2.20 (s, 3H), 3.70 (s, 3H), 3.99 (s, 2H), 4.55 (s,2H), 5.19 (s, 2H), 6.06 (s, H), 7.77 (s, H), 8.17 (s, H), MS M+Hcalculated for 384.2; found 384.2. Melting point: 145-146° C.

Example 10-911-benzyl-4-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-2-ethyl-1,2,4-triazolidine-3,5-dione

1-benzyl-4-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-1,2,4-triazolidine-3,5-dione(100 mg, 0.27 mmol), bromoethane (149 mg, 1.36 mmol), and cesiumcarbonate (355 mg, 1.1 mmol) in DMF (5 mL) were heated at 80° C. for 15hours. The reaction was cooled to room temperature, diluted with brine(50 mL) and extracted with ethyl acetate (2×, 50 mL). The combinedorganic extracts were dried over magnesium sulfate, filtered andconcentrated on the rotovap. The residue was purified by HPLC (5 to 95%acetonitrile in H₂O: 25 minute gradient) to afford1-benzyl-4-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-2-ethyl-1,2,4-triazolidine-3,5-dione(40 mg, 37%) as an oil. ¹H NMR (DMSO-d₆, 400 MHz): δ0.97 (t, J=7.2 Hz,3H), 2.13 (s, 3H), 2.39 (s, 3H), 3.58 (q, J=6.8 Hz, 2H), 4.80 (s, 2H),5.18 (s, 2H), 7.28-7.36 (m, 5H), 7.77 (s, 1H), 8.19 (s, 1H). MS M+Hcalculated 395.18; found 395.2. The title compound was shown to inhibithT2R08 bitter receptor and had an IC₅₀ of 0.04 μM.

Example 10-91a1-benzyl-4-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-1,2,4-triazolidine-3,5-dione

1-benzyl-N-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)hydrazinecarboxamide(2.00 g, 5.88 mmol), ethyl chloroformate (6.38 g, 58.77 mmol), andtriethylamine (1.78 g, 17.63 mmol) in acetonitrile (50 mL) were heatedat 100° C. for 48 hours. The mixture was cooled to 80° C., 1 M NaOH(aq)(5 mL) was added and the reaction mixture was stirred for 1 hour. Thereaction was cooled to room temperature and concentrated on the rotpvap.The residue was dissolved in dichloromethane and filtered to removesalts and the solution was concentrated to afford1-benzyl-4-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-1,2,4-triazolidine-3,5-dione(1.28 g, 60%) as a yellow oil. MS M+H calculated 367.14; found 367.2.

Example 10-91b1-benzyl-N-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)hydrazinecarboxamide

1-((3,5-dimethylisoxazole-4-yl)methyl-1H-pyrazole-4-carbonyl azide (2.79g, 11.33 mmol) in toluene (70 mL) was heated at reflux for 4 hours toafford 4-((4-isocyanato-1H-pyrazol-1-yl)methyl)-3,5-dimethylisoxazole insitu. Benzylhydrazine dihydrochloride (2.44 g, 12.45 mmol) andtriethylamine (2.29 g, 22.64 mmol) were added to the mixture. Themixture was heated at 100° C. for an additional 4 hours. The reactionwas cooled to room temperature, diluted with ethyl acetate (150 mL) andfiltered through celite. The mother liquor was then washed with brine(150 mL) and the organic layer were dried over magnesium sulfate,filtered and concentrated. The residue was purified by silica columnchromotography (100% to 90% dichloromethane in methanol: 30 minutegradient) to afford1-benzyl-N-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)hydrazinecarboxamide(2.00 g, 52%) as an oil. MS M+H calculated 341.16; found 341.2.

Example 10-921-benzyl-4-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-2-(2-methoxyethyl)-1,2,4-triazolidine-3,5-dione

Prepared as in example 10-91 from1-benzyl-4-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-1,2,4-triazolidine-3,5-dione(example 10-91a) and 2-bromoethyl methyl ether. Yield: 20%. MS M+Hcalculated 425.19; found 425.2. The title compound was shown to inhibithT2R08 bitter receptor and had an IC₅₀ of 0.06 μM.

Example 10-931-benzyl-4-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-2-propyl-1,2,4-triazolidine-3,5-dione

Prepared as in example 10-91 from1-benzyl-4-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-1,2,4-triazolidine-3,5-dione(example 10-91a) and 1-bromopropane. Yield: 38%. MS M+H calculated409.19; found 409.2. The title compound was shown to inhibit hT2R08bitter receptor and had an IC₅₀ of 0.06 μM.

Example 10-943-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-1-(3-phenylpropyl)imidazolidine-2,4-dione

Prepared as in example 10-52 from3-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)imidazolidine-2,4-dione(example 10-1) and (3-bromopropyl)benzene. Yield: 36%. MS M+H calculated394.2; found 394.2. The title compound was shown to inhibit hT2R08bitter receptor and had an IC₅₀ of 0.26 μM.

Example 10-951-benzyl-2-butyl-4-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl-1,2,4-triazolidine-3,5-dione

Prepared as in example 10-91 from1-benzyl-4-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-1,2,4-triazolidine-3,5-dione(example 10-91a) and 1-bromobutane. Yield: 22%. MS M+H calculated423.21; found 423.15. The title compound was shown to inhibit hT2R08bitter receptor and had an IC₅₀ of 0.41 μM.

Example 10-96 tert-butyl3-((3-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-2,4-dioxoimidazolidin-1-yl)methyl)benzylcarbamate

3-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)imidazolidine-2,4-dione(example 10-1) (070 mg, 0.254 mmol), tert-butyl3-(hydroxymethyl)benzylcarbamate (0.254 mmol, 60 mg), diethylazodicarboxylate (0.50 mmol, 86 mg), and P-tBu₃ (125 mL, 0.50 mmol) werestirred in THF (1 mL) for 4 hours. The reaction was diluted with ethylacetate (1.5 mL) and washed with saturated sodium bicarbonate solution(2×, 1.5 mL). The organic phase was collected and the mixture wasconcentrated under a stream of nitrogen. The crude product was purifiedby column chromatography on silica gel using ethyl acetate as theeluent. The pure fractions were combined and the solvents were removedon the rotovap to afford tert-butyl3-((3-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-2,4-dioxoimidazolidin-1-yl)methyl)benzylcarbamateas a white, solid (112 mg, 90%). NMR (CDCl₃, 400 MHz): δ 1.44 (s, 9H),2.19 (s, 3H), 2.42 (s, 3H), 3.48 (bs, 1H), 3.84 (s, 2H), 4.31-4.30 (d,J=6 Hz, 1H), 4.60 (s, 2H), 4.87 (bs, 1H), 5.06 (s, 2H), 7.36-7.16 (m,4H), 7.92 (s, 1H), 8.08 (s, 1H). The title compound was shown to inhibithT2R08 bitter receptor and had an IC₅₀ of 0.90 μM.

Example 10-973-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-5-(hydroxymethyl)imidazolidine-2,4-dione

4-((4-isocyanato-1H-pyrazol-1-yl)methyl)-3,5-dimethylisoxazole (example10-1) (784 mg, 3.6 mmol), serine methyl ester hydrochoride (672 mg, 4.32mmol) and triethylamine (1 mL, 7.2 mmol) were refluxed in toluene (16mL) for 8 hours. The reaction was allowed to cool to room temperatureand then the solution was concentrated on the rotovap. The product waspurified by reverse phase HPLC (5 to 95% acetonitrile in H₂O: 16 minutegradient) to afford3-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-5-(hydroxymethyl)imidazolidine-2,4-dioneas a white solid (60 mg, 25%). ¹H NMR (CDCl₃, 400 MHz): δ(s, 3H), 2.40(s, 3H), 3.13-3.07 (m, 1H), 3.94-3.93 (d, J=4 Hz, 2H), 4.21-4.19 (t, J=4Hz, 1H), 5.03 (s, 2H), 6.68 (bs, 1H), 7.87 (s, 1H), 7.99 (s, 1H). Thetitle compound was shown to inhibit hT2R08 bitter receptor and had anIC₅₀ of 3 μM.

Compound hT2R8 No. Compound IC₅₀ (μM) 10-66

4-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-1-(4-fluorophenethyl)-2-methyl-1,2,4-triazolidine-3,5-dione 0.012 10-15

3-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H- pyrazol-4-yl)-1-(2-(methylthio)benzyl)imidazolidine-2,4-dione 0.016 10-42

5-benzyl-3-(1-((3,5-dimethylisoxazol-4- yl)methyl)-1H-pyrazol-4-yl)-1-methylimidazolidine-2,4-dione 0.017 10-26

3-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H- pyrazol-4-yl)-1-(3-methylbenzyl)imidazolidine-2,4-dione 0.019 10-43

1-benzyl-3-(1-((3,5-dimethylisoxazol-4- yl)methyl)-1H-pyrazol-4-yl)-5-methylimidazolidine-2,4-dione 0.020 10-89

1-benzyl-4-(1-((3,5-dimethylisoxazol-4- yl)methyl)-1H-pyrazol-4-yl)-2-(methoxymethyl)-1,2,4-triazolidine-3,5-dione 0.024 10-10

3-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H- pyrazol-4-yl)-1-(3-hydroxybenzyl)imidazolidine-2,4-dione 0.026 10-84

3-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H- pyrazol-4-yl)-1-(4-fluorophenethyl)imidazolidine-2,4-dione 0.027 10-91

1-benzyl-4-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-2-ethyl-1,2,4- triazolidine-3,5-dione 0.04110-12

3-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H- pyrazol-4-yl)-1-(4-hydroxybenzyl)imidazolidine-2,4-dione 0.043 10-79

3-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H- pyrazol-4-yl)-1-(4-methoxyphenethyl)imidazolidine-2,4-dione 0.044 10-31

3-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-1-(4-fluorobenzyl)imidazolidine- 2,4-dione 0.045 10-32

4-((3-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-2,4-dioxoimidazolidin-1- yl)methyl)benzonitrile 0.04810-35

1-(3,4-dimethoxybenzyl)-3-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4- yl)imidazolidine-2,4-dione0.051 10-36

1-(benzo[d][1,3]dioxol-5-ylmethyl)-3-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4- yl)imidazolidine-2,4-dione0.053 10-92

1-benzyl-4-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-2-(2-methoxyethyl)-1,2,4-triazolidine-3,5-dione 0.057 10-27

3-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-1-(3-fluorobenzyl)imidazolidine- 2,4-dione 0.058 10-18

3-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H- pyrazol-4-yl)-1-(2-methoxybenzyl)imidazolidine-2,4-dione 0.060 10-93

1-benzyl-4-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-2-propyl-1,2,4- triazolidine-3,5-dione 0.06210-5

3-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H- pyrazol-4-yl)-1-(2-phenoxyethyl)imidazolidine-2,4-dione 0.063 10-77

3-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H- pyrazol-4-yl)-1-(3-fluorophenethyl)imidazolidine-2,4-dione 0.064 10-30

3-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H- pyrazol-4-yl)-1-(4-methylbenzyl)imidazolidine-2,4-dione 0.065 10-94

3-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H- pyrazol-4-yl)-1-(3-phenylpropyl)imidazolidine-2,4-dione 0.068 10-11

3-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H- pyrazol-4-yl)-1-(2-hydroxybenzyl)imidazolidine-2,4-dione 0.069 10-6

3-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H- pyrazol-4-yl)-1-(3-methoxybenzyl)imidazolidine-2,4-dione 0.073 10-23

3-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-1-(2-nitrobenzyl)imidazolidine- 2,4-dione 0.081 10-76

3-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H- pyrazol-4-yl)-1-(2-methoxyphenethyl)imidazolidine-2,4-dione 0.097 10-13

3-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-1-(3-hydroxy-4- methoxybenzyl)imidazolidine-2,4-dione0.087 10-21

3-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-1-(2-fluorobenzyl)imidazolidine- 2,4-dione 0.090 10-7

methyl 3-((3-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-2,4- dioxoimidazolidin-1-yl)methyl)benzoate0.102 10-4

(R)-5-benzyl-3-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)imidazolidine-2,4-dione 0.112 10-3

(S)-5-benzyl-3-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)imidazolidine-2,4-dione 0.113 10-2

5-benzyl-3-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)imidazolidine-2,4-dione 0.117 10-16

3-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H- pyrazol-4-yl)-1-(2-(2-methoxyethoxy)benzyl)imidazolidine-2,4-dione 0.131 10-56

3-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H- pyrazol-4-yl)-5-(3-methoxybenzyl)imidazolidine-2,4-dione 0.131 10-20

3-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H- pyrazol-4-yl)-1-(2-methylbenzyl)imidazolidine-2,4-dione 0.133 10-87

1-(3,4-dimethoxyphenethyl)-3-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4- yl)imidazolidine-2,4-dione0.137 10-78

3-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-1-phenethylimidazolidine-2,4-dione 0.139 10-9

3-((3-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-2,4-dioxoimidazolidin-1- yl)methyl)-N-methylbenzamide0.141 10-85

3-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H- pyrazol-4-yl)-1-(3-methoxyphenethyl)imidazolidine-2,4-dione 0.158 10-34

1-(2-aminobenzyl)-3-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4- yl)imidazolidine-2,4-dione0.163 10-24

3-((3-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-2,4-dioxoimidazolidin-1- yl)methyl)benzaldehyde 0.20110-90

1-((1,3-dimethyl-1H-pyrazol-5-yl)methyl)-3-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)imidazolidine-2,4-dione 0.215 10-22

3-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H- pyrazol-4-yl)-1-(2-(trifluoromethyl)benzyl)imidazolidine-2,4-dione 0.221 10-75

3-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-1-((6-methoxypyridin-2- yl)methyl)imidazolidine-2,4-dione0.229 10-53

1-benzyl-3-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)imidazolidine-2,4-dione 0.257 10-81

1-(2-chlorophenethyl)-3-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4- yl)imidazolidine-2,4-dione0.248 10-74

1-(cyclohexylmethyl)-3-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4- yl)imidazolidine-2,4-dione0.276 10-58

5-(cyclopentylmethyl)-3-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4- yl)imidazolidine-2,4-dione0.316 10-28

1-((1,5-dimethyl-1H-pyrazol-3-yl)methyl)-3-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)imidazolidine-2,4-dione 0.322 10-33

3-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H- pyrazol-4-yl)-1-(3-(hydroxymethyl)benzyl)imidazolidine-2,4-dione 0.322 10-59

3-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H- pyrazol-4-yl)-5-(4-hydroxybenzyl)imidazolidine-2,4-dione 0.327 10-38

3-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-1-((1-methyl-1H-pyrazol-3-yl)methyl)imidazolidine-2,4-dione 0.363 10-46

3-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H- pyrazol-4-yl)-5-(3-phenylpropyl)imidazolidine-2,4-dione 0.484 10-95

1-benzyl-2-butyl-4-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-1,2,4- triazolidine-3,5-dione 0.409 10-17

3-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H- pyrazol-4-yl)-1-(2-(methylsulfinyl)benzyl)imidazolidine-2,4-dione 0.412 10-86

3-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H- pyrazol-4-yl)-1-(4-hydroxyphenethyl)imidazolidine-2,4-dione 0.412 10-83

3-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H- pyrazol-4-yl)-1-(2-fluorophenethyl)imidazolidine-2,4-dione 0.442 10-55

1-((3,5-dimethylisoxazol-4-yl)methyl)-3-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)imidazolidine-2,4-dione 0.479 10-60

5-(3,4-dihydroxybenzyl)-3-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4- yl)imidazolidine-2,4-dione0.513 10-19

2-((3-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-2,4-dioxoimidazolidin-1- yl)methyl)benzonitrile 0.55010-54

3-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H- pyrazol-4-yl)-1-(pyridin-2-ylmethyl)imidazolidine-2,4-dione 0.609 10-71

3-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-1-((6-(hydroxymethyl)pyridin-2-yl)methyl)imidazolidine-2,4-dione 0.721 10-48

2-(1-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-2,5-dioxoimidazolidin-4-yl)- N-phenylacetamide 0.74710-47

5-(benzyloxymethyl)-3-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4- yl)imidazolidine-2,4-dione0.766 10-96

tert-butyl 3-((3-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-2,4- dioxoimidazolidin-1-yl)methyl)benzylcarbamate 0.891 10-61

3-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-5-isobutylimidazolidine-2,4-dione 0.912 10-80

3-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-1-(2-(naphthalen-1- yl)ethyl)imidazolidine-2,4-dione 0.92710-57

5-(cyclohexylmethyl)-3-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4- yl)imidazolidine-2,4-dione0.962 10-51

3-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-1-methylimidazolidine-2,4-dione 0.966 10-82

1-(3-chlorophenethyl)-3-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4- yl)imidazolidine-2,4-dione0.982 10-45

3-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-5-phenethylimidazolidine-2,4-dione 0.793 10-72

1-((3,4-dimethoxypyridin-2-yl)methyl)-3-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)imidazolidine-2,4-dione 0.999 10-25

3-((3-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-2,4-dioxoimidazolidin-1- yl)methyl)benzonitrile 1.00310-37

1-(3-((dimethylamino)methyl)benzyl)-3-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)imidazolidine-2,4-dione 1.220 10-41

3-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-5-methylimidazolidine-2,4-dione 1.285 10-49

N-benzyl-2-(1-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-2,5- dioxoimidazolidin-4-yl)acetamide 1.32910-73

3-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-1-((tetrahydrofuran-2- yl)methyl)imidazolidine-2,4-dione1.362 10-62

3-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-5-isopropylimidazolidine-2,4-dione 1.440 10-1

3-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)imidazolidine-2,4-dione 1.696 10-50

2-(1-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-2,5-dioxoimidazolidin-4-yl)-N-(3-methoxybenzyl)acetamide 1.773 10-8

3-((3-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-2,4-dioxoimidazolidin-1- yl)methyl)benzoic acid 1.7989-5

1-benzyl-3-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)imidazolidine- 2,4,5-trione 2.493 10-29

3-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H- pyrazol-4-yl)-1-(4-methoxybenzyl)imidazolidine-2,4-dione 3.117 10-63

(E)-5-benzylidene-3-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4- yl)imidazolidine-2,4-dione10-67

4-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-1-methyl-2-(2-phenoxyethyl)- 1,2,4-triazolidine, 3,5-dione10-97

3-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H- pyrazol-4-yl)-5-(hydroxymethyl)imidazolidine-2,4-dione 9-8

1-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-3-phenethyl-1,3,5-triazinane- 2,4,6-trione 6.151 9-7

5-benzyl-1-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-3-phenethyl- 1,3,5-triazinan-2-one 6.580 9-9

1-benzyl-3-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-1,3,5-triazinane- 2,4,6-trione 1.562 10-88

1-benzyl-3-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)imidazolidin-2-one 1.250 9-5

1-benzyl-3-(1-((3,5-dimethylisoxazol-4- yl)methyl)-1H-pyrazol-4-yl)tetrahydropyrimidin-2(1H)-one 3.434 9-6

1-benzyl-3-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-1,3,5-triazinan-2-one 6.029 10-68

4-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-1,2,4-triazolidine-3,5-dione 10-45

3-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-5-phenethylimidazolidine-2,4-dione 0.793 10-44

2-(1-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-2,5-dioxoimidazolidin-4- yl)acetic acid 10-65

1-benzyl-4-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-2-methyl-1,2,4- triazolidine-3,5-dione 0.01910-70

1,2-dibenzyl-4-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-1,2,4-triazolidine- 3,5-dione 1.799 10-69

4-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-1,2-dimethyl-1,2,4-triazolidine- 3,5-dione 0.707 10-52

3-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-1,5,5-trimethylimidazolidine-2,4-dione 0.810 10-14

3-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H- pyrazol-4-yl)-1-(3-(2-methoxyethoxy)benzyl)imidazolidine-2,4-dione 0.071

Example 11 hT2R8 Contributes to the Bitterness of Saccharin for Peoplewith the “Non-Taster” Versions of the hT2R43 and hT2R44 Genes

FIG. 6 shows the dose-response relationships and the effects ofsaccharin on receptor activities in transfected cells expressingvariants of hT2R43, hT2R44 and hT2R8. hT2R8 is less responsive tosaccharin in the in vitro assay than the “taster” hT2R43-W35 andhT2R44-W35 alleles, but responds better than the “non-taster” hT2R43-S35and hT2R44-R35 alleles. Pronin et al., Curr. Biol. 17: 1403-8 (2007).Five individuals with the “taster” alleles (hT2R43-W35 and/orhT2R44-W35) and five with the “non-taster” alleles (hT2R43-S35 andhT2R44-R35) were selected based on genotyping analysis. Each subject waspresented with 6 pairs of solutions and asked to determine which of thesamples in a pair tasted more bitter. The result shown in Table Table 8,below, shows that hT2R8 blocker Cpd-D reduces bitter taste of saccharinfor people with the “non-taster” alleles of hT2R43 and hT2R44 but has noeffect on people with the “taster” alleles of those genes.

TABLE 8 Taste Test Results Selected as Taste Bitter Genotype more bitterTest agonist group without +Cpd-D P value 1 saccharin hT2R43-W35 and/or13 17 0.82 hT2R44-W35 2 saccharin hT2R43-S35 and 27 3 <0.001 hT2R44-R35

Example 12 Substituted Hydantoin Analogs (T2R8 Blockers) Example 12-13-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-1-(3-hydroxybenzyl)-5,5-dimethylimidazolidine-2,4-dione

1-(3-(tert-butyldimethylsilyloxy)benzyl)-3-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-5,5-dimethylimidazolidine-2,4-dione(Example 12-1a) (2.00 g, 3.82 mmol) was dissolved in methanol (200 mL)and treated with HCl (2.0 M solution in diethyl ether, 10 equivalents).The solution was stirred for 16 hours at 50° C. then allowed to cooldown to room temperature and the solvent was removed under reducedpressure. The residue was dissolved in ethanol and recrystallized at 4°C. to give3-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-1-(3-hydroxybenzyl)-5,5-dimethylimidazolidine-2,4-dione(1.50 g, 3.67 mmol, 96%) as a white solid. ¹H NMR (DMSO-d₆, 400 MHz):

1.31 (s, 6H), 2.16 (s, 3H), 2.42 (s, 3H), 4.47 (s, 2H), 5.20 (s, 2H),6.65 (ddd, J=0.8, 2.4, 8 Hz, 1H), 6.79 (m, 2H), 7.11 (t, J=8 Hz, 1H),7.83 (d, J=0.8 Hz, 1H), 8.21 (d, J=0.8 Hz, 1H), 9.36 (s, H). MS 410(MH⁺). The title compound was shown to inhibit hT2R08 bitter receptorand had an IC₅₀ of 0.05 μM

Example 12-1a1-(3-(tert-butyldimethylsilyloxy)benzyl)-3-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-5,5-dimethylimidazolidine-2,4-dione

1-((3,5-dimethylisoxazol-4-yl) methyl)-1H-pyrazole-4-carbonyl azide(Example 10-1a) (1.26 g, 5.12 mmol) along with molecular sieves arestirred at reflux in dry toluene (100 mL) under nitrogen until noemission of nitrogen was observed, which indicates complete conversionof 1-((3,5-dimethylisoxazol-4-yl) methyl)-1H-pyrazole-4-carbonyl azideinto 4-((4-isocyanato-1H-pyrazol-1-yl)methyl)-3,5-dimethylisoxazole. Asolution of ethyl2-(3-(tert-butyldimethylsilyloxy)benzylamino)-2-methylpropanoate(Example 12-1b) (1.50 g, 4.27 mmol) in dry toluene (50 mL) was prepared.To this solution, the mixture obtained above, which contains4-((4-isocyanato-1H-pyrazol-1-yl)methyl)-3,5-dimethylisoxazole andmolecular sieves, was added in one portion at 50° C. and refluxed for 24h. The mixture was allowed to cool down to room temperature, filteredover a pad of celite and evaporated. The residue was further purifiedover silica gel using ethyl acetate/hexanes as eluent and the solventswere evaporated to provide a colorless oily material (2.10 g, 94%). MS524 (MH⁺).

Example 12-1b Ethyl2-(3-(tert-butyldimethylsilyloxy)benzylamino)-2-methylpropanoate

To 3-(tert-butyldimethylsilyloxy)benzaldehyde (Example 12-1c) (3.00 g,12.7 mmol) in DCE (100 mL) was added ethyl 2-amino-2-methylpropanoatehydrochloride (25 mmol, ˜2 equiv.), molecular sieves 4A (5 g), and Et₃N(2 equiv.). The mixture was stirred for 6 hours at room temperature andtreated with NaBH(OAc)₃ (2 equiv.). After the addition was completed,the reaction mixture was stirred overnight under nitrogen. The mixturewas diluted with DMC (200 mL), carefully quenched with saturated aqueousNaHCO₃ solution and the layers were separated. The aqueous phase wasfurther extracted with DCM (2×100 mL). The combined organic extract waswashed with water (50 mL), brine (100 mL) and dried over Na₂SO₄. Thesolvent was removed under vacuum and the residue was purified oversilica gel using hexanes/EtOAc (2/8) to give ethyl2-(3-(tert-butyldimethylsilyloxy)benzylamino)-2-methylpropanoate (4.02g, 90%). MS 352 (MH⁺)

Example 12-1c 3-(tert-butyldimethylsilyloxy)benzaldehyde

3-Hydroxyldehyde (19.35 g, 81.88 mmol) was dissolved in anhydrous DCM(300 mL) and immidazole (3 equiv.) was added. The mixture was cooleddown to 0° C. and a solution of tert-butylchlorodimethylsilane (1.5equiv.) in DCM (200 mL) was added dropwise. After stirring overnight atroom temperature, the reaction mixture was washed with 1M aqueous HClsolution, brine and dried over Na₂SO₄. After filtration, the solvent wasremoved under reduced pressure to provide the desired productquantitative yield. MS 237 (MH⁺).

Example 12-23-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-1-(3-hydroxy-4-methoxybenzyl)-5,5-dimethylimidazolidine-2,4-dione

Prepared as in Example 12-1 from1-(3-(tert-butyldimethylsilyloxy)-4-methoxybenzyl)-3-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-5,5-dimethylimidazolidine-2,4-dione(Example 12-2a). Yield 60%, white solid. ¹H NMR (DMSO-d₆, 400 MHz): δ1.29 (s, 6H), 2.16 (s, 3H), 2.42 (s, 3H), 3.73 (s, 3H), 4.42 (s, 2H),5.20 (s, 2H), 6.82 (m, 3H) 7.83 (s, 1H), 8.20 (s, 1H), 8.94 (br s, 1H).MS 440 (MH⁺). The title compound was shown to inhibit hT2R08 bitterreceptor and had an IC₅₀ of 0.06 μM

Example 12-2a1-(3-(tert-butyldimethylsilyloxy)-4-methoxybenzyl)-3-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-5,5-dimethylimidazolidine-2,4-dione

Prepared as in Example 12-1a from2-(3-(tert-butyldimethylsilyloxy)-4-methoxybenzylamino)-2-methylpropanoate(Example 12-2b) and1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazole-4-carbonyl azide(Example 10-1a). Yield 85%, white solid. MS 554 (MH⁺).

Example 12-2b Ethyl2-(3-(tert-butyldimethylsilyloxy)-4-methoxybenzylamino)-2-methylpropanoate

Prepared as in Example 12-1b from3-(tert-butyldimethylsilyloxy)-4-methoxybenzaldehyde (Example 12-2c) andethyl 2-amino-2-methylpropanoate hydrochloride. Yield 94%, colorlessliquid. MS 382 (MH⁺).

Example 12-2c 3-(tert-butyldimethylsilyloxy)-4-methoxybenzaldehyde

Prepared as in Example 12-1c from 3-hydroxy-4-methoxybenzaldehyde andtert-butylchlorodimethylsilane. Yield 100%, yellowish liquid. MS 267(MH⁺).

Example 12-31-(3,5-bis(tert-butyldimethylsilyloxy)benzyl)-3-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-5,5-dimethylimidazolidine-2,4-dione

Prepared as in Example 12-1 from1-(3,5-bis(tert-butyldimethylsilyloxy)benzyl)-3-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-5,5-dimethylimidazolidine-2,4-dione(Example 12-3a). Yield 92%, white solid. ¹H NMR (DMSO-d₆, 400 MHz):

₁□

s, 6H), 2.17 (s, 3H), 2.43 (s, 3H), 4.38 (s, 2H), 5.21 (s, 2H), 6.10 (m,1H), 6.23 (m, 2H), 7.84 (d, J=0.4 Hz, 1H), 8.22 (d, J=0.4 Hz, 1H), 9.20(br s, 1H), 9.21 (br, s, 1H). Mp 213-215° C. MS 426 (MH⁺). The titlecompound was shown to inhibit hT2R08 bitter receptor and had an IC₅₀ of0.01 μM

Example 12-3a1-(3,5-bis(tert-butyldimethylsilyloxy)benzyl)-3-(1-(3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-5,5-dimethylimidazolidine-2,4-dione

Prepared as in Example 12-1a from ethyl2-(3,5-bis(tert-butyldimethylsilyloxy)benzylamino)-2-methylpropanoate(Example 12-3b) and1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazole-4-carbonyl azide(Example 10-1a). Yield 82%, white solid. MS 654 (MH⁺).

Example 12-3b ethyl2-(3,5-bis(tert-butyldimethylsilyloxy)benzylamino)-2-methylpropanoate

Prepared as in Example 12-1b from3,5-bis(tert-butyldimethylsilyloxy)benzaldehyde (Example 12-3c) andethyl 2-amino-2-methylpropanoate hydrochloride. Yield 88%, colorlessliquid. MS 482 (MH⁺).

Example 12-3c 3,5-bis(tert-butyldimethylsilyloxy)benzaldehyde

Prepared as in Example 12-1c from 3,5-dihydroxybenzaldehyde andtert-butylchlorodimethylsilane. Yield 100%, yellowish liquid. MS 267(MH⁺).

Example 12-47-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-5-(3-hydroxybenzyl)-5,7-diazaspiro[3.4]octane-6,8-dione

Prepared as in Example 12-1 from5-(3-(tert-butyldimethylsilyloxy)benzyl)-7-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-5,7-diazaspiro[3.4]octane-6,8-dione(Example 12-4a). Yield 74%, white solid. ¹H NMR (DMSO-d₆, 400 MHz):

1.73 (m, 1H), 1.98 (m, 1H), 2.15 (s, 3H), 2.27 (m, 2H) 2.42 (s, 3H),2.45 (m, 2H), 4.65 (s, 2H), 5.20 (s, 2H), 6.65 (m, 1H), 6.73 (m, 1H),6.76 (m, 1H), 7.13 (t, J=8 Hz, 1H), 7.82 (d, J=0.8 Hz, 1H), 8.20 (d,J=0.8 Hz, 1H), 9.39 (s, H). MS 422 (MH⁺). The title compound was shownto inhibit hT2R08 bitter receptor and had an IC₅₀ of 0.03 μM

Example 12-4a5-(3-(tert-butyldimethylsilyloxy)benzyl)-7-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-5,7-diazaspiro[3.4]octane-6,8-dione

Prepared as in Example 12-1a from ethyl1-(3-(tert-butyldimethylsilyloxy)benzylamino)cyclobutanecarboxylate(Example 12-4b) and1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazole-4-carbonyl azide(Example 10-1a) Yield 89%. MS 536 (MH⁺).

Example 12-4b ethyl 1-(3-(tert-butyldimethylsilyloxy)benzylamino)cyclobutanecarboxylate

Prepared as in Example 12-1b from3-(tert-butyldimethylsilyloxy)benzaldehyde (Example 12-1c) and ethyl1-aminocyclobutanecarboxylate hydrochloride. Yield 77%, colorlessliquid. MS 364 (MH⁺).

Example 12-53-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-1-(3-hydroxybenzyl)-1,3-diazaspiro[4.4]nonane-2,4-dione

Prepared as in Example 12-1 from1-(3-(tert-butyldimethylsilyloxy)benzyl)-3-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-1,3-diazaspiro[4.4]nonane-2,4-dione(Example 12-5a). Yield 81%, white solid. ¹H NMR (DMSO-d₆, 400 MHz):

1.72 (m, 4H), 1.89 (m, 4H), 2.16 (s, 3H), 2.42 (s, 3H), 4.49 (s, 2H),5.20 (s, 2H), 6.65 (m, 1H), 6.76 (m, 2H), 7.12 (t, J=8.0 Hz, 1H), 7.84(d, J=0.4 Hz, 1H), 8.21 (d, J=0.4 Hz, 1H), 9.38 (s, H). MS 436 (MH⁺).The title compound was shown to inhibit hT2R08 bitter receptor and hadan IC₅₀ of 0.07 μM

Example 12-5a1-(3-(tert-butyldimethylsilyloxy)benzyl)-3-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-1,3-diazaspiro[4.4]nonane-2,4-dione

Prepared as in Example 12-1a from ethyl1-(3-(tert-butyldimethylsilyloxy)benzylamino)cyclobutanecarboxylate(Example 12-5b) and1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazole-4-carbonyl azide(Example 10-1a). Yield 89%. MS 550 (MH⁺).

Example 12-5b Ethyl1-(3-(tert-butyldimethylsilyloxy)benzylamino)-cyclopentanecarboxylate

Prepared as in Example 1b-JF from3-(tert-butyldimethylsilyloxy)benzaldehyde (Example 12-1c) and ethyl1-aminocyclopentanecarboxylate hydrochloride. Yield 77%, a colorlessliquid. MS 378 (MH⁺).

Example 12-6(S)-3-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-1-(3-hydroxybenzyl)-5-methylimidazolidine-2,4-dione

Prepared as in Example 12-1 from(S)-1-(3-(tert-butyldimethylsilyloxy)benzyl)-3-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-5-methylimidazolidine-2,4-dione(Example 12-6a). Yield 93%, white solid. ¹H NMR (DMSO-d₆, 400 MHz): ¹HNMR (DMSO-D6, 400 MHz):

(d, J=7.2 Hz, 3H), 2.15 (s, 3H), 2.41 (s, 3H), 4.10 (q, J=7.2 Hz, 1H),4.34 (d, J=15.6, Hz, 1H), 4.60 (d, J=15.6 Hz, 1H), 5.20 (s, 2H), 6.72(m, 3H), 7.13 (t, J=8.0 Hz, 1H), 7.81 (d, J=0.4 Hz, 1H), 8.19 (d, J=0.4Hz, 1H), 9.41 (br s, 1H). MS 396 (MH⁺). The title compound was shown toinhibit hT2R08 bitter receptor and had an IC₅₀ of 0.02 μM

Example 12-6a(S)-1-(3-(tert-butyldimethylsilyloxy)benzyl)-3-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-5-methylimidazolidine-2,4-dione

Prepared as in Example 12-1a from (S)-ethyl2-(3-(tert-butyldimethylsilyloxy)benzylamino)propanoate (Example 12-6b)and 1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazole-4-carbonyl azide(Example 10-1a). Yield 75%. MS 510 (MH⁺).

Example 12-6b (S)-methyl2-(3-(tert-butyldimethylsilyloxy)benzylamino)-propanoate

Prepared as in Example 12-1b from3-(tert-butyldimethylsilyloxy)benzaldehyde (Example 12-1c) and(S)-methyl 2-aminopropanoate hydrochloride. Yield 80%, colorless liquid.MS 324 (MH⁺).

Example 12-7(R)-3-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-1-(3-hydroxybenzyl)-5-methylimidazolidine-2,4-dione

Prepared as in Example 12-1 from(R)-1-(3-(tert-butyldimethylsilyloxy)benzyl)-3-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-5-methylimidazolidine-2,4-dione(Example 12-7a). Yield 96%, white solid. ¹H NMR (DMSO-d₆, 400 MHz): ¹HNMR (DMSO-D6, 400 MHz):

(d, J=7.2 Hz, 3H), 2.15 (s, 3H), 2.41 (s, 3H), 4.10 (q, J=7.2 Hz, 1H),4.34 (d, J=15.6, Hz, 1H), 4.60 (d, J=15.6 Hz, 1H), 5.20 (s, 2H), 6.72(m, 3H), 7.13 (t, J=8.0 Hz, 1H), 7.81 (d, J=0.4 Hz, 1H), 8.19 (d, J=0.4Hz, 1H), 9.41 (br s, 1H). MS 396 (MH⁺). The title compound was shown toinhibit hT2R08 bitter receptor and had an IC₅₀ of 0.02 μM

Example 12-7a(R)-1-(3-(tert-butyldimethylsilyloxy)benzyl)-3-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-5-methylimidazolidine-2,4-dione

Prepared as in Example 12-1a from (R)-ethyl2-(3-(tert-butyldimethylsilyloxy)benzylamino)propanoate (Example 12-7b)and 1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazole-4-carbonyl azide(Example 10-1a). Yield 77%. MS 510 (MH⁺).

Example 12-7b (R)-methyl2-(3-(tert-butyldimethylsilyloxy)benzylamino)-propanoate

Prepared as in Example 12-1b from3-(tert-butyldimethylsilyloxy)benzaldehyde (Example 12-1c) and(R)-methyl 2-aminopropanoate hydrochloride. Yield 82%, colorless liquid.MS 324 (MH⁺).

Example 12-8(S)-3-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-1-(3-hydroxybenzyl)-5-isopropylimidazolidine-2,4-dione

Prepared as in Example 12-1 from(S)-1-(3-(tert-butyldimethylsilyloxy)benzyl)-3-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-5-isopropylimidazolidine-2,4-dione(Example 12-8a). Yield 50%, white solid. MS 423 (MH⁺). The titlecompound was shown to inhibit hT2R08 bitter receptor and had an IC₅₀ of0.65 μM

Example 12-8a(S)-1-(3-(tert-butyldimethylsilyloxy)benzyl)-3-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-5-isopropylimidazolidine-2,4-dione

Prepared as in Example 12-1a from (S)-ethyl2-(3-(tert-butyldimethylsilyloxy)benzylamino)-3-methylbutanoate (Example12-8b) and 1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazole-4-carbonylazide (Example 10-1a). MS 538 (MH⁺).

Example 12-8b (S)-ethyl2-(3-(tert-butyldimethylsilyloxy)benzylamino)-3-methylbutanoate

Prepared as in Example 12-1b from3-(tert-butyldimethylsilyloxy)benzaldehyde (Example 12-1c) and (S)-ethyl2-amino-3-methylbutanoate hydrochloride. Yield 97%, colorless liquid. MS352 (MH⁺).

Example 12-9(S)-3-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-1-(3-hydroxybenzyl)-5-(hydroxymethyl)imidazolidine-2,4-dione

Prepared as in Example 12-1 from(S)-1-(3-(tert-butyldimethylsilyloxy)benzyl)-5-((tert-butyldimethylsilyloxy)methyl)-3-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)imidazolidine-2,4-dione(Example 12-9a). Yield 72%, white solid. ¹H NMR (DMSO-d₆, 400 MHz): 2.13(s, 3H), 2.40 (s, 3H), 3.78 (m, 2H), 4.01 (t, J=2.4 Hz, 1H), 4.80 (d,J=15.6, Hz, 1H), 5.18 (s, 2H), 5.20 (br.t, J=5.6 Hz, 1H), 6.66 (m, 3H),6.72 (m, 3H), 7.11 (t, J=8.0 Hz, 1H), 7.80 (d, J=0.8 Hz, 1H), 8.18 (d,J=0.8 Hz, 1H), 9.38 (br s, 1H). MS 412 (MH⁺). The title compound wasshown to inhibit hT2R08 bitter receptor and had an IC₅₀ of 0.04 μM

Example 12-9a(S)-1-(3-(tert-butyldimethylsilyloxy)benzyl)-5-((tert-butyldimethylsilyloxy)methyl)-3-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)imidazolidine-2,4-dione

Prepared as in Example 12-1a from (S)-methyl3-(tert-butyldimethylsilyloxy)-2-(3-(tert-butyldimethylsilyloxy)benzylamino)propanoate(Example 12-9b) and1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazole-4-carbonyl azide(Example 10-1a). Yield 65%. MS 640 (MH⁺).

Example 12-9b (S)-methyl3-(tert-butyldimethylsilyloxy)-2-(3-(tert-butyldimethylsilyloxy)benzylamino)propanoate

Prepared as in Example 12-1c from (S)-methyl2-(3-(tert-butyldimethylsilyloxy)benzylamino)-3-hydroxypropanoate(Example 12-9c). Yield 85%, colorless liquid. MS 454 (MH⁺).

Example 12-9c (S)-methyl2-(3-(tert-butyldimethylsilyloxy)benzylamino)-3-hydroxypropanoate

To a solution of 3-(tert-butyldimethylsilyloxy)benzaldehyde (Example12-1c) 1.52 g (6.43 mmol), triethyl amine (5 mL) and (S)-methyl2-amino-3-hydroxypropanoate hydrochloride (1.00 g, 6.43 mmol) in DCM (30mL) was added MgSO₄ and the solution was stirred at RT for 36 h. MgSO₄was then filtered off and DCM was removed under vacuum. The residue wasdissolved in dry MeOH (40 mL), cooled to 0° C. and treated in smallportions with NaBH₄ (1.2 equiv.). The mixture was stirred at 0° C. for 3h, quenched with water (10 mL) and concentrated by removing MeOH. Theresidue was extracted with Ethyl acetate to provide (S)-methyl2-(3-(tert-butyldimethylsilyloxy)benzylamino)-3-hydroxypropanoate asyellowish oil (2.10 g, 96%). MS 340 (MH⁺).

Example 12-103-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-1-(3-hydroxybenzyl)-5-(hydroxymethyl)-5-methylimidazolidine-2,4-dione

Prepared as in Example 12-1 from1-(3-(tert-butyldimethylsilyloxy)benzyl)-5-((tert-butyldimethylsilyloxy)methyl)-3-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-5-methylimidazolidine-2,4-dione(Example 12-10a). Yield 62%, white solid. MS 426 (MH⁺). The titlecompound was shown to inhibit hT2R08 bitter receptor and had an IC₅₀ of0.13 μM

Example 12-10a1-(3-(tert-butyldimethylsilyloxy)benzyl)-5-((tert-butyldimethylsilyloxy)methyl)-3-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-5-methylimidazolidine-2,4-dione

Prepared as in Example 12-1a from methyl3-(tert-butyldimethylsilyloxy)-2-(3-(tert-butyldimethylsilyloxy)benzylamino)-2-methylpropanoate(Example 12-10b) and1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazole-4-carbonyl azide(Example 10-1a). Yield 79%. MS 654 (MH⁺).

Example 12-10b methyl3-(tert-butyldimethylsilyloxy)-2-(3-(tert-butyldimethylsilyloxy)benzylamino)-2-methylpropanoate

Prepared as in Example 12-1c from methyl2-(3-(tert-butyldimethylsilyloxy)benzylamino)-3-hydroxy-2-methylpropanoate(Example 12-10c). Yield 76%, colorless liquid. MS 468 (MH⁺).

Example 12-10c methyl2-(3-(tert-butyldimethylsilyloxy)benzylamino)-3-hydroxy-2-methylpropanoate

Prepare as in Example 12-9c from3-(tert-butyldimethylsilyloxy)benzaldehyde (Example 12-1c) and methyl2-amino-3-hydroxy-2-methylpropanoate hydrochloride. Yield 97%, yellowishoil. MS 354 (MH⁺).

Example 12-11(S)-1-benzyl-3-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-5-methylimidazolidine-2,4-dione

Prepared as in Example 12-1a from (S)-methyl 2-(benzylamino)propanoate(Example 12-11a) and1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazole-4-carbonyl azide(Example 10-1a). Yield 89. MS 380 (MH⁺). The title compound was shown toinhibit hT2R08 bitter receptor and had an IC₅₀ of 0.01 μM

Example 12-11a (S)-methyl 2-(benzylamino)propanoate

Prepared as in Example 12-1b from benzaldehyde and (R)-methyl2-aminopropanoate hydrochloride. Yield 95%. MS 194 (MH⁺).

Example 12-126-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-4-(3-hydroxy-4-methoxybenzyl)-4,6-diazaspiro[2.4]heptane-5,7-dione

Prepared as in Example 12-1 from4-(3-(tert-butyldimethylsilyloxy)benzyl)-6-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-4,6-diazaspiro[2.4]heptane-5,7-dione(Example 12-12a). Yield 90%, white solid. MS 440 (MH⁺). The titlecompound was shown to inhibit hT2R08 bitter receptor and had an IC₅₀ of0.02 μM

Example 12-12a4-(3-(tert-butyldimethylsilyloxy)-4-methoxybenzyl)-6-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-4,6-diazaspiro[2.4]heptane-5,7-dione

1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazole-4-carbonyl azide(Example 10-1a) (650 mg, 2.63 mmol) along with molecular sieves arestirred at reflux in dry toluene (50 mL) under nitrogen gas until noemission of nitrogen was observed. This solution was added to a solutionof methyl1-(3-(tert-butyldimethylsilyloxy)-4-methoxybenzylamino)cyclopropanecarboxylate(Example 12-12b) (800 mg, 2.19 mmol) in dry toluene (25 mL) in oneportion at 50° C. and stirred at the same temperature for 24 h. Themixture was directly filtered over a pad of celite. The filtrate wasconcentrated and the residue was dissolved in dry MeOH (50 mL). Tendrops of NH₃ solution in MeOH were added and the mixture stirred at RTfor 2 h. Volatiles were then removed under reduce pressure and theresidue purified over silica gel using ethyl acetate/hexanes as eluent(3/7) to give the desired product as a colorless oil (856 mg, 75%). MS522 (MH⁺).

Example 12-12b methyl1-(3-(tert-butyldimethylsilyloxy)-4-methoxybenzylamino)cyclopropanecarboxylate

Prepared as in Example 12-1b from3-(tert-butyldimethylsilyloxy)-4-methoxybenzaldehyde (Example 12-2c) andmethyl 1-aminocyclopropanecarboxylate hydrochloride. Yield 79%. MS 366(MH⁺).

Example 12-134-(3,4-dimethoxybenzyl)-6-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-4,6-diazaspiro[2.4]heptane-5,7-dione

Prepared as in Example 12-12a from methyl1-(3,4-dimethoxybenzylamino)cyclopropanecarboxylate (Example 12-13a) and1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazole-4-carbonyl azide(Example 10-1a). Yield 88%. ¹H NMR (DMSO-d₆, 400 MHz):

1.21 (m, 2H), 1.41 (m, 2H), 2.16 (s, 3H), 2.42 (s, 3H), 3.28 (s, 3H),3.726 (s, 3H), 3.728 (s, 3H), 4.39 (s, 2H), 5.21 (s, 2H), 6.82 (dd,J=2.0 Hz, 8.0 Hz, 1H), 6.90 (m, 2H), 7.83 (d, J=0.4 Hz, 1H), 8.22 (d,J=0.4 Hz, 1H). MS 452 (MH⁺). The title compound was shown to inhibithT2R08 bitter receptor and had an IC₅₀ of 0.02 μM

Example 12-13a methyl1-(3,4-dimethoxybenzylamino)cyclopropanecarboxylate

Prepared as in Example 12-1b from 3,4-dimethoxybenzaldehyde and methyl1-aminocyclopropanecarboxylate hydrochloride. Yield 82%. MS 266 (MH⁺).

Example 12-146-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-4-(3-(methoxymethyl)benzyl)-4,6-diazaspiro[2.4]heptane-5,7-dione

Prepared as in Example 12-12a from methyl1-(3-(methoxymethyl)benzylamino)cyclopropanecarboxylate (Example 12-14a)and 1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazole-4-carbonyl azide(Example 10-1a). Yield 81%. 1H NMR (DMSO-D6, 400 MHz):

1.22 (m, 2H), 1.38 (m, 2H), 2.16 (s, 3H), 2.42 (s, 3H), 3.28 (s, 3H),4.39 (s, 2H), 4.47 (s, 2H), 5.21 (s, 2H), 7.22 (m, 3H), 7.33 (t, J=7.6Hz, 1H), 7.83 (d, J=0.8 Hz, 1H), 8.22 (d, J=0.8 Hz, 1H). MS 436 (MH+).The title compound was shown to inhibit hT2R08 bitter receptor and hadan IC₅₀ of 0.03 μM

Example 12-14a methyl1-(3-(methoxymethyl)benzylamino)cyclopropanecarboxylate

Prepared as in Example 12-1b from 3-(methoxymethyl)benzaldehyde (Example12-14b) and methyl 1-aminocyclopropanecarboxylate hydrochloride. Yield82%. MS 250 (MH⁺).

Example 12-14b 3-(methoxymethyl)benzaldehyde

3-(bromomethyl)benzaldehyde (1.00 g, 5.02 mmol) was dissolved in MeOH(100 mL) and treated with 0.5M solution of MeONa (5 equiv.) in MeOH. Themixture was stirred overnight at RT and MeOH was evaporated underreduced pressure. The residue was acidified to pH ˜6 with 1M aqueous HClsolution. DCM (100 mL) was added and the mixture was vigorously stirredovernight. The organic layer was separated, dried over Na2SO4 and thesolvent was removed under reduce pressure to furnish the desiredmaterial as yellowish oil (560 mg, 3.75 mmol, 74%). ¹H NMR (DMSO, 400MHz):

3.33 (s, 3H), 4.51 (s, 1H), 7.62 (m, 2H), 7.85 (m, 2H), 10.02 (s, 1H).MS 151 (MH⁺).

Example 12-156-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-4-(3-hydroxybenzyl)-4,6-diazaspiro[2.4]heptane-5,7-dione

Prepared as in Example 12-1 from4-(3-(tert-butyldimethylsilyloxy)benzyl)-6-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-4,6-diazaspiro[2.4]heptane-5,7-dione(Example 12-15a). Yield 70%, white solid. ¹H NMR (DMSO-d₆, 400 MHz):

1.21 (m, 2H), 1.38 (m, 2H), 2.14 (s, 3H), 2.40 (s, 3H), 4.36 (s, 2H),5.19 (s, 2H), 6.65 (m, 3H), 7.11 (dt, J=1.2, 7.6 Hz, 1H), 7.81 (d, J=0.8Hz, 1H), 8.21 (d, J=0.8 Hz, 1H), 9.41 (s, H). [MS 408 (MH⁺). The titlecompound was shown to inhibit hT2R08 bitter receptor and had an IC₅₀ of0.02 μM

Example 12-15a4-(3-(tert-butyldimethylsilyloxy)benzyl)-6-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-4,6-diazaspiro[2.4]heptane-5,7-dione

Prepared as in Example 12-1a from ethyl1-(3-(tert-butyldimethylsilyloxy)benzylamino)cyclopropanecarboxylate(Example 12-15b) and1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazole-4-carbonyl azide(Example 10-1a). Yield 15%. MS 522 (MH⁺).

Example 12-15b ethyl1-(3-(tert-butyldimethylsilyloxy)benzylamino)-cyclopropanecarboxylate

Prepared as in Example 12-1b from3-(tert-butyldimethylsilyloxy)benzaldehyde (Example 12-1c) and ethyl1-aminocyclopropanecarboxylate hydrochloride. Yield 87%, colorlessliquid. MS 350 (MH⁺).

Example 12-164-((3-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-5,5-dimethyl-2,4-dioxoimidazolidin-1-yl)methyl)benzonitrile

3-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-5,5-dimethylimidazolidine-2,4-dione(Example 12-16a) (250 mg, 0.83 mmol), 4-(chloromethyl)benzonitrile (126mg, 0.83 mmol), and cesium carbonate (536 mg, 1.65 mmol) were stirred indimethylformamide (4 mL) at 80° C. for 5 hours. The reaction was allowedto cool to room temperature and then filtered. The filtrate was dilutedwith ethyl acetate (4 mL) and then water (2 mL) was added. The organicphase was collected, dried over anhydrous Na2SO4, and then concentratedunder reduced pressure. The residue was purified by reverse phase HPLC(gradient 10% to 100% methanol in water) and further recrystallized fromethanol. Yield 30%. 1H NMR (DMSO-d6, 400 MHz):

□

s, 6H), 2.15 (s, 3H), 2.42 (s, 3H), 4.65 (s, 2H), 5.20 (s, 2H), 7.60 (d,J=8.4 Hz, 2H), 7.81 (m, 3H), 8.20 (d, J=0.8 Hz, 1H). MS 419 (MH+). Thetitle compound was shown to inhibit hT2R08 bitter receptor and had anIC₅₀ of 0.04 μM

Example 12-16a3-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-5,5-dimethylimidazolidine-2,4-dione

1-((3,5-dimethylisoxazol-4-yl) methyl)-1H-pyrazole-4-carbonyl azide(Example 10-1a) (10 g, 41.0 mmol) is stirred under nitrogen in drytoluene (150 mL) along with molecular sieves at reflux until no emissionof nitrogen was observed, which indicates complete conversion of1-((3,5-dimethylisoxazol-4-yl) methyl)-1H-pyrazole-4-carbonyl azide into4-((4-isocyanato-1H-pyrazol-1-yl)methyl)-3,5-dimethylisoxazole. Thesolution was cooled down to 50° C. and ethyl 2-amino-2-methylpropanoatehydrochloride (5.30 g, 41.0 mmol) was added in one portion. Stirringcontinued for 3 hours at 50° C. followed by reflux for 12 hours. Themixture was filtered over a pad of celite. The filtrate was concentratedon the rotovap and the residue was purified over silica gel using 25%ethyl acetate in hexanes. Yield 65% (8.00 g), white solid. 1H NMR(DMSO-d6, 400 MHz):

□

s, 6H), 2.15 (s, 3H), 2.41 (s, 3H), 5.18 (s, 2H), 7.78 (d, J=0.4 Hz,1H), 8.16 (d, J=0.4 Hz, 1H), 8.59 (br s, 1H). MS 304 (MH+). Thiscompound was shown to inhibit hT2R08 bitter receptor and had an IC₅₀ of2.27 μM.

Example 12-173-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-1-(3-hydroxy-5-methoxybenzyl)-5,5-dimethylimidazolidine-2,4-dione

1-(3,5-dimethoxybenzyl)-3-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-5,5-dimethylimidazolidine-2,4-dione(Example 12-18) (114 mg, 0.252 mmol) was dissolved in dichloromethane (1mL). The mixture was cooled down to −78° C. and boron tribromide (1.0 Msolution in dichloromethane, 1.25 mL) was added dropwise. After completeaddition, the mixture was allowed to warm to room temperature andstirring continued for 3 hours. The mixture was quenched with water andthe organic phase was separated, dried over anhydrous Na₂SO₄, filteredand evaporated under reduced pressure. The residue was suspended inmethanol (1 mL) and purified by reverse phase HPLC (25 minute gradient:acetonitrile/water). Yield 47% (45 mg), white solid. NMR (DMSO-d₆, 400MHz):

□

s, 6H), 2.16 (s, 3H), 2.42 (s, 3H), 3.67 (s, 3H), 4.43 (s, 2H), 5.20 (s,2H), 6.22 (t, J=2.4 Hz, 1H), 6.39 (m, 2H), 7.83 (d, J=0.4 Hz, 1H), 8.21(s, 1H), 9.40 (br s, 1H). Mp. 72-74° C. MS 440 (MH⁺). The title compoundwas shown to inhibit hT2R08 bitter receptor and had an IC₅₀ of 0.02 μM.

Example 12-181-(3,5-dimethoxybenzyl)-3-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-5,5-dimethylimidazolidine-2,4-dione

Prepared as in Example 12-16 from3-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-5,5-dimethylimidazolidine-2,4-dione(Example 12-16a) and 1-(chloromethyl)-3,5-dimethoxybenzene. MS 454(MH⁺). The title compound was shown to inhibit hT2R08 bitter receptorand had an IC₅₀ of 0.3 μM.

Example 12-193-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-1-(4-fluoro-3-hydroxybenzyl)-5,5-dimethylimidazolidine-2,4-dione

Prepared as in Example 12-17 from3-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-1-(4-fluoro-3-methoxybenzyl)-5,5-dimethylimidazolidine-2,4-dione(Example 12-20). Yield 30%, white solid. MS 428 (MH⁺). The titlecompound was shown to inhibit hT2R08 bitter receptor and had an IC₅₀ of0.01 μM.

Example 12-203-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-1-(4-fluoro-3-methoxybenzyl)-5,5-dimethylimidazolidine-2,4-dione

Prepared as in Example 12-16 from3-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-5,5-dimethylimidazolidine-2,4-dione(Example 12-16a) and 4-(chloromethyl)-1-fluoro-2-methoxybenzene. MS 442(MH⁺). The title compound was shown to inhibit hT2R08 bitter receptorand had an IC₅₀ of 0.18 μM.

Example 12-211-((3,5-dimethylisoxazol-4-yl)methyl)-3-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-5,5-dimethylimidazolidine-2,4-dione

Prepared as in Example 12-16 from3-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-5,5-dimethylimidazolidine-2,4-dione(Example 12-16a) and 4-(chloromethyl)-3,5-dimethylisoxazole. MS 413(MH⁺). The title compound was shown to inhibit hT2R08 bitter receptorand had an IC₅₀ of 0.08 μM.

Example 12-223-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-5,5-dimethyl-1-(2-methylbenzyl)imidazolidine-2,4-dione

Prepared as in Example 12-16 from3-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-5,5-dimethylimidazolidine-2,4-dione(Example 12-16a) and 1-(bromomethyl)-2-methylbenzene. MS 408 (MH⁺). Thetitle compound was shown to inhibit hT2R08 bitter receptor and had anIC₅₀ of 0.1 μM.

Example 12-233-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-1-(4-methoxyphenethyl)-5,5-dimethylimidazolidine-2,4-dione

Prepared as in Example 12-16 from3-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-5,5-dimethylimidazolidine-2,4-dione(Example 12-16a) and 1-(2-bromoethyl)-4-methoxybenzene. MS 438 (MH⁺).The title compound was shown to inhibit hT2R08 bitter receptor and hadan IC₅₀ of 0.12 μM.

Example 12-243-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-5,5-dimethyl-1-(1-phenylethyl)imidazolidine-2,4-dione

Prepared as in Example 16 from3-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-5,5-dimethylimidazolidine-2,4-dione(Example 16a) and (1-bromoethyl)benzene. MS 408 (MH⁺). The titlecompound was shown to inhibit hT2R08 bitter receptor and had an IC₅₀ of0.15 μM.

Example 12-251-((1,5-dimethyl-1H-pyrazol-3-yl)methyl)-3-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-5,5-dimethylimidazolidine-2,4-dione

Prepared as in Example 12-16 from3-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-5,5-dimethylimidazolidine-2,4-dione(Example 12-16a) and 3-(chloromethyl)-1,5-dimethyl-1H-pyrazole. MS 412(MH⁺). The title compound was shown to inhibit hT2R08 bitter receptorand had an IC₅₀ of 0.16 μM.

Example 12-261-((1,3-dimethyl-1H-pyrazol-5-yl)methyl)-3-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-5,5-dimethylimidazolidine-2,4-dione

Prepared as in Example 12-16 from3-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-5,5-dimethylimidazolidine-2,4-dione(Example 12-16a) and 5-(chloromethyl)-1,3-dimethyl-1H-pyrazole. MS 412(MH+). The title compound was shown to inhibit hT2R08 bitter receptorand had an IC₅₀ of 0.23 μM.

Example 12-273-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-5,5-dimethyl-1-(2-phenoxyethyl)imidazolidine-2,4-dione

Prepared as in Example 12-16 from3-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-5,5-dimethylimidazolidine-2,4-dione(Example 12-16a) and (2-bromoethoxy)benzene. MS 424 (MH⁺). The titlecompound was shown to inhibit hT2R08 bitter receptor and had an IC₅₀ of0.35 μM.

Example 12-283-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-1-(2-methoxyethyl)-5,5-dimethylimidazolidine-2,4-dione

Prepared as in Example 12-16 from3-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-5,5-dimethylimidazolidine-2,4-dione(Example 12-16a) and 1-bromo-2-methoxyethane. MS 362 (MH⁺). The titlecompound was shown to inhibit hT2R08 bitter receptor and had an IC₅₀ of0.59 μM.

Example 12-293-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-1-(4-fluorobenzyl)-5,5-dimethylimidazolidine-2,4-dione

Prepared as in Example 12-16 from3-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-5,5-dimethylimidazolidine-2,4-dione(Example 12-16a) and 1-(bromomethyl)-4-fluorobenzene. Yield 40%, whitesolid. 1H NMR (DMSO-d6, 400 MHz):

s, 6H), 2.14 (s, 3H), 2.40 (s, 3H), 4.53 (s, 2H), 5.18 (s, 2H), 7.13 (m,2H) 7.43 (m, 2H), 7.81 (s, H), 8.18 (s, 1H). MS 412 (MH+). The titlecompound was shown to inhibit hT2R08 bitter receptor and had an IC50 of0.08 μM.

Example 12-301-(3,4-dimethoxybenzyl)-3-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-5,5-dimethylimidazolidine-2,4-dione

Prepared as in Example 12-16 from3-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-5,5-dimethylimidazolidine-2,4-dione(Example 12-16a) and 4-(bromomethyl)-1,2-dimethoxybenzene. Yield 67%,white solid. 1H NMR (DMSO-d6, 400 MHz):

s, 6H), 2.16 (s, 3H), 2.42 (s, 3H), 3.72 (s, 3H), 3.73 (s, 3H), 4.49 (s,2H), 5.20 (s, 2H) 6.92 (m, 2H), 7.00 (d, J=2.0 Hz, 1H), 7.83 (d, J=0.4Hz, 1H), 8.20 (d, J=0.4 Hz, 1H). MS 454 (MH+). The title compound wasshown to inhibit hT2R08 bitter receptor and had an IC₅₀ of 0.08 μM.

Example 12-313-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-1-(2-methoxybenzyl)-5,5-dimethylimidazolidine-2,4-dione

Prepared as in Example 12-16 from3-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-5,5-dimethylimidazolidine-2,4-dione(Example 12-16a) and 1-(bromomethyl)-2-methoxybenzene. Yield 53%, whitesolid. 1H NMR (DMSO-d6, 400 MHz):

s, 6H), 2.16 (s, 3H), 2.42 (s, 3H), 3.83 (s, 3H), 4.50 (s, 2H), 5.20 (s,2H), 6.91 (dt, J=1.2, 7.6 Hz, 1H) 7.00 (dd, J=0.8, 8.0 Hz, 1H), 7.27 (m,2H), 7.82 (d, J=0.8 Hz, 1H), 8.20 (d, J=0.8 Hz, 1H). MS 424 (MH+). Thetitle compound was shown to inhibit hT2R08 bitter receptor and had anIC₅₀ of 0.08 μM.

Example 12-323-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-1-(3-methoxybenzyl)-5,5-dimethylimidazolidine-2,4-dione

Prepared as in Example 12-16 from3-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-5,5-dimethylimidazolidine-2,4-dione(Example 12-16a) and 1-(bromomethyl)-3-methoxybenzene. Yield 30%, whitesolid. ¹H NMR (DMSO-d₆, 400 MHz):

₁s, 6H), 2.16 (s, 3H), 2.42 (s, 3H), 3.74 (s, 3H), 4.53 (s, 2H), 5.20(s, 2H), 6.83 (m, 1H) 6.96 (m, 2H), 7.25 (t, J=8.0 Hz, 1H), 7.83 (d,J=0.8 Hz, 1H), 8.21 (d, J=0.8 Hz, 1H). mp 107-108° C. MS 424 (MH⁺). Thetitle compound was shown to inhibit hT2R08 bitter receptor and had anIC₅₀ of 0.08 μM.

Example 12-333-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-1-(4-methoxybenzyl)-5,5-dimethylimidazolidine-2,4-dione

Prepared as in Example 12-16 from3-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-5,5-dimethylimidazolidine-2,4-dione(Example 12-16a) and 1-(bromomethyl)-4-methoxybenzene. Yield 35%,colorless gel (195 mg, 35%). 1H NMR (DMSO-d6, 400 MHz):

□

s, 6H), 2.14 (s, 3H), 2.40 (s, 3H), 3.71 (s, 3H), 4.48 (s, 2H), 5.18 (s,2H) 6.87 (d, J=8.8 Hz, 2H), 7.31 (d, J=8.4 Hz, 2H), 7.80 (s, 1H), 8.18(s, 1H). MS 424 (MH+). The title compound was shown to inhibit hT2R08bitter receptor and had an IC₅₀ of 0.11 μM.

Example 12-343-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-1-(3-(hydroxymethyl)benzyl)-5,5-dimethylimidazolidine-2,4-dione

Prepared as in Example 12-16 from3-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-5,5-dimethylimidazolidine-2,4-dione(Example 12-16a) and (3-(bromomethyl)phenyl)methanol. Yield 70%, whitesolid. ¹H NMR (DMSO-d₆, 400 MHz): δ1.29 (s, 6H), 2.14 (s, 3H), 2.40 (s,3H), 4.47 (d, J=6.0 Hz, 2H), 4.54 (s, 2H), 5.15 (t, J=5.6 Hz, 1H) 5.2(s, 2H), 7.25 (m, 3H), 7.32 (s, 1H), 7.82 (s, 1H) 8.20 (s, 1H). MS 424(MH⁺). The title compound was shown to inhibit hT2R08 bitter receptorand had an IC₅₀ of 0.11 μM.

Example 12-353-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-1-(3-(methoxymethyl)benzyl)-5,5-dimethylimidazolidine-2,4-dione

To a solution of3-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-1-(3-(hydroxymethyl)benzyl)-5,5-dimethylimidazolidine-2,4-dione(Example 12-34) (559 mg, 1.32 mmol) in anhydrous dimethylformamide (10mL) at 0° C., was added sodium hydride (53 mg, 1.32 mmol), followed byiodomethane (99 ul, 1.58 mmol). The reaction was allowed to warm to roomtemperature and stirred for about 4 hours. The resulting mixture wasdiluted with ethyl acetate (40 mL) and ice water (10 mL), The organicphase was collected, dried over anhydrous MgSO₄, filtered and thenconcentrated on the rotovap. The residue was purified by reverse phaseHPLC (gradient 10% to 100% methanol/water) and further purified bycolumn chromatography (60% EtOAc in hexane). Yield 36%, colorless gel.¹H NMR (DMSO-d₆, 400 MHz): δ1.29 (s, 6H), 2.14 (s, 3H), 2.40 (s, 3H),3.25 (s, 3H), 4.37 (s, 2H), 4.55 (s, 2H), 5.18 (s, 2H), 7.19 (t, 1H)7.30 (d, 2H), 7.32 (s, 1H), 7.82 (s, 1H) 8.20 (s, 1H). MS 438 (MH⁺). Thetitle compound was shown to inhibit hT2R08 bitter receptor and had anIC₅₀ of 0.13 μM.

Example 12-363-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-5,5-dimethyl-1-(pyridin-3-ylmethyl)imidazolidine-2,4-dione

Prepared as in Example 12-1a from ethyl2-methyl-2-(pyridin-3-ylmethylamino)propanoate (Example 12-36a) and1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazole-4-carbonyl azide(Example 10-1a). Yield 47%, white a solid. ¹H NMR (CDCl₃, 400 MHz):

1.36 (s, 6H), 2.18 (s, 3H), 2.41 (s, 3H), 4.57 (s, 2H), 5.04 (s, 2H),7.27 (m, 1H), 7.74 (m, 1H), 7.94 (d, J=0.4 Hz, 1H), 8.09 (d, J=0.4 Hz,1H), 8.54 (br. d, J=4.0 Hz, 1H), 8.59 (br. S, 1H). MS 395 (MH⁺). Thetitle compound was shown to inhibit hT2R08 bitter receptor and had anIC₅₀ of 0.22 μM.

Example 12-36a ethyl 2-methyl-2-(pyridin-3-ylmethylamino)propanoate

Prepared as in Example 12-1b from nicotinaldehyde and ethyl1-aminocyclopropanecarboxylate hydrochloride. Yield 95%, a white solid.MS 223 (MH⁺).

Example 12-37(R)-1-benzyl-3-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-5-methylimidazolidine-2,4-dione

Prepared as in Example 12-1a from (R)-methyl 2-(benzylamino)propanoate(Example 12-37a) and1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazole-4-carbonyl azide(Example 10-1a). Yield 89%. MS 380 (MH⁺). The title compound was shownto inhibit hT2R08 bitter receptor and had an IC₅₀ of 0.01 μM.

Example 12-37a (R)-methyl 2-(benzylamino)propanoate

Prepared as in Example 12-1b from benzaldehyde and (R)-methyl2-aminopropanoate hydrochloride Yield 95%. MS 194 (MH⁺).

Additional compounds were synthesized according to the above describedprocedures and experimentally tested and were found to have relativelyhigh effectiveness as inhibitors of hT2R8 bitter receptors. The resultsof that testing are shown in the table below.

Compound hT2R8 No. Compound IC₅₀ (μM) 12-38

3-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-1-(4-hydroxybenzyl)-5,5-dimethylimidazolidine-2,4-dione 0.07 12-39

3-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-1-(2-hydroxybenzyl)-5,5-dimethylimidazolidine-2,4-dione 0.07 12-40

3-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-1-((6-methoxypyridin-2-yl)methyl)-5,5-dimethylimidazolidine-2,4-dione 0.08 12-41

3-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-1-((6-methoxypyridin-2-yl)methyl)-5,5-dimethylimidazolidine-2,4-dione 0.08 12-42

3-((3-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-5,5-dimethyl- 2,4-dioxoimidazolidin-1-yl)methyl)benzonitrile 0.09 12-43

1-(2,3-dihydroxybenzyl)-3-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-5,5-dimethylimidazolidine-2,4-dione 0.10 12-44

1-(2,6-difluorobenzyl)-3-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-5,5-dimethylimidazolidine-2,4-dione 0.10 12-45

3-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-1-(2-fluorobenzyl)-5,5- dimethylimidazolidine-2,4-dione0.11 12-46

3-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-1-(2-fluorobenzyl)-5,5- dimethylimidazolidine-2,4-dione0.12 12-47

3-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-1-(2-fluorobenzyl)-5,5- dimethylimidazolidine-2,4-dione0.12 12-48

3-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-5,5-dimethyl-1-(3- methylbenzyl)imidazolidine-2,4-dione0.12 12-49

1-(3,4-dimethoxyphenethyl)-3-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-5,5-dimethylimidazolidine-2,4-dione 0.13 12-50

3-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-1-(3-fluorophenethyl)-5,5-dimethylimidazolidine-2,4-dione 0.15 12-51

3-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-1-((5-methoxypyridin-2-yl)methyl)-5,5-dimethylimidazolidine-2,4-dione 0.19 12-52

3-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-5,5-dimethyl-1-(pyridin-4-ylmethyl)imidazolidine-2,4-dione 0.19 12-53

3-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-1-(2-fluorophenethyl)-5,5-dimethylimidazolidine-2,4-dione 0.20 12-54

1-(2,5-dimethoxybenzyl)-3-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-5,5-dimethylimidazolidine-2,4-dione 0.20 12-55

methyl 3-((3-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-5,5-dimethyl-2,4-dioxoimidazolidin-1-yl)methyl)benzoate 0.22 12-56

3-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-5,5-dimethyl-1- phenethylimidazolidine-2,4-dione 0.2312-57

methyl 4-((3-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-5,5-dimethyl-2,4-dioxoimidazolidin-1-yl)methyl)benzoate 0.23 12-58

3-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-1-(2-methoxyphenethyl)-5,5-dimethylimidazolidine-2,4-dione 0.24 12-59

1-(2,5-difluorobenzyl)-3-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-5,5-dimethylimidazolidine-2,4-dione 0.24 12-60

3-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-1-(3-methoxyphenethyl)-5,5-dimethylimidazolidine-2,4-dione 0.25 12-61

3-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-5,5-dimethyl-1-(pyridin-2-ylmethyl)imidazolidine-2,4-dione 0.26 12-62

1-((2,3-dihydrobenzo[b][1,4]dioxin-6-yl)methyl)-3-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-5,5- dimethylimidazolidine-2,4-dione 0.2712-63

3-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-1-(3-(2-hydroxyethoxy)benzyl)-5,5-dimethylimidazolidine-2,4-dione 0.28 12-64

1-(2,3-dimethoxybenzyl)-3-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-5,5-dimethylimidazolidine-2,4-dione 0.30 12-65

3-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-1-(2-fluoro-6-methoxybenzyl)-5,5-dimethylimidazolidine-2,4-dione 0.31 12-66

1-(2,3-difluorobenzyl)-3-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-5,5-dimethylimidazolidine-2,4-dione 0.31 12-67

1-benzyl-3-(1-((3,5-dimethylisoxazol-4- yl)methyl)-1H-pyrazol-4-yl)-5,5-dimethylimidazolidine-2,4-dione 0.32 12-68

1-benzyl-3-(1-((3,5-dimethylisoxazol-4- yl)methyl)-1H-pyrazol-4-yl)-5,5-dimethylimidazolidine-2,4-dione 0.33 12-69

3-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-1-(4-fluorophenethyl)-5,5-dimethylimidazolidine-2,4-dione 0.33 12-70

1-(3,5-difluorophenethyl)-3-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-5,5-dimethylimidazolidine-2,4-dione 0.40 12-71

3-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-5,5-dimethyl-1-((1- methyl-1H-pyrazol-3-yl)methyl)imidazolidine-2,4-dione 0.42 12-72

3-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-1-((6-hydroxypyridin-2-yl)methyl)-5,5-dimethylimidazolidine-2,4-dione 0.44 12-73

3-((3-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-5,5-dimethyl-2,4-dioxoimidazolidin-1-yl)methyl)benzoic acid 0.46 12-74

1-(2,4-difluorobenzyl)-3-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-5,5-dimethylimidazolidine-2,4-dione 0.53 12-75

methyl 2-(3-((3-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-5,5- dimethyl-2,4-dioxoimidazolidin-1-yl)methyl)phenoxy)acetate 0.53 12-76

3-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-1-(4-methoxy-3-methylbenzyl)-5,5-dimethylimidazolidine-2,4-dione 0.54 12-77

3-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-1-(2-hydroxyethyl)-5,5- dimethylimidazolidine-2,4-dione0.56 12-78

3-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-5,5-dimethyl-1-(3- phenylpropyl)imidazolidine-2,4-dione0.58 12-79

4-((3-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-5,5-dimethyl-2,4-dioxoimidazolidin-1-yl)methyl)benzonitrile 0.05 12-80

3-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-1-(3-(2-methoxyethoxy)benzyl)-5,5-dimethylimidazolidine-2,4-dione 0.71 12-81

3-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-5,5-dimethyl-1-(4- methylbenzyl)imidazolidine-2,4-dione0.74 12-82

3-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-1-(2-fluoro-3-methylbenzyl)-5,5-dimethylimidazolidine-2,4-dione 0.81 12-83

1-(3,4-dimethylbenzyl)-3-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-5,5-dimethylimidazolidine-2,4-dione 1.22 12-84

3-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-5,5-dimethyl-1-(3-(trifluoromethyl)benzyl)imidazolidine-2,4-dione 1.54 12-85

1-(2,6-dimethylbenzyl)-3-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-5,5-dimethylimidazolidine-2,4-dione 1.58 12-86

1-(2,4-dimethylbenzyl)-3-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-5,5-dimethylimidazolidine-2,4-dione 1.89 12-87

1-(3-(2-(dimethylamino)ethoxy)benzyl)-3-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-5,5-dimethylimidazolidine-2,4-dione 2.08 12-88

3-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-1-(4-(hydroxymethyl)benzyl)-5,5-dimethylimidazolidine-2,4-dione 0.36 12-89

4-((3-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-5,5-dimethyl-2,4-dioxoimidazolidin-1-yl)methyl)benzoic acid 2.31 12-90

2-(3-((3-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-5,5-dimethyl-2,4-dioxoimidazolidin-1-yl)methyl)phenoxy)acetic acid 2.85 12-91

2-((3-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-5,5-dimethyl-2,4-dioxoimidazolidin-1-yl)methyl)benzoic acid 4.23 12-92

3-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-1-(4-(methoxymethyl)benzyl)-5,5-dimethylimidazolidine-2,4-dione 0.28

Example 13 Constrained Hydantoin Analogs (T2R8 Blockers) Example 13-1(S)-2-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-7-hydroxy-10,10a-dihydroimidazo[1,5-b]isoquinoline-1,3(2H,5H)-dione

Prepared as in Example 12-1 from(S)-7-(tert-butyldimethylsilyloxy)-2-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-10,10a-dihydroimidazo[1,5-b]isoquinoline-1,3(2H,5H)-dione (Example 13-1a). Yield 98%, white solid. ¹H NMR (DMSO-d₅, 400MHz): δ 2.15 (s, 3H), 2.41 (s, 3H), 2.85 (dd, J=8.0, 15.2 Hz, 1H), 3.07(dd, J=4.8, 15.2 Hz, 1H), 4.33 (m, 2H), 4.79 (d, J=16.8 Hz, 1H), 5.21(s, 2H), 6.64 (m, 2H), 7.06 (d, J=78.0 Hz, 1H), 7.81 (d, J=0.8 Hz, 1H),8.20 (d, J=0.8 Hz, 1H), 9.39 (s, H). [MS 394 (MH⁺). The title compoundwas shown to inhibit hT2R08 bitter receptor and had an IC₅₀ of 0.02 μM.

Example 13-1a(S)-7-(tert-butyldimethylsilyloxy)-2-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-10,10a-dihydroimidazo[1,5-b]isoquinoline-1,3(2H,5H)-dione

Prepared as in Example 12-1a from (S)-methyl7-(tert-butyldimethylsilyloxy)-1,2,3,4-tetrahydroisoquinoline-3-carboxylate(Example 13-1b) and1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazole-4-carbonyl azide(Example 10-1a). Yield 92%, yellow solid. MS 508 (MH⁺).

Example 13-1b (S)-methyl7-(tert-butyldimethylsilyloxy)-1,2,3,4-tetrahydroisoquinoline-3-carboxylate

Prepared as in Example 12-1c from (S)-methyl7-hydroxy-1,2,3,4-tetrahydroisoquinoline-3-carboxylate hydrochloride(Example 13-1c) and tert-butylchlorodimethylsilane. Yield 87%, yellowoil. MS 322 (MH⁺).

Example 13-1c (S)-methyl7-hydroxy-1,2,3,4-tetrahydroisoquinoline-3-carboxylate hydrochloride

Prepared by refluxing(S)-7-hydroxy-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid in aconcentrated methanolic solution of HCl. Yield 100%, Beige powder. MS208 (MH⁺—Cl⁻).

Example 13-2(R)-2-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-7-hydroxy-10,10a-dihydroimidazo[1,5-b]isoquinoline-1,3(2H,5H)-dione

Prepared as in Example 12-1 from(R)-7-(tert-butyldimethylsilyloxy)-2-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-10,10a-dihydroimidazo[1,5-b]isoquinoline-1,3(2H,5H)-dione (Example 13-2a). Yield 85%, white solid. [MS 394 (MH⁺). Thetitle compound was shown to inhibit hT2R08 bitter receptor and had anIC₅₀ of 0.03 μM

Example 13-2a(R)-7-(tert-butyldimethylsilyloxy)-2-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-10,10a-dihydroimidazo[1,5-b]isoquinoline-1,3(2H,5H)-dione

Prepared as in Example 12-1a from (R)-methyl7-(tert-butyldimethylsilyloxy)-1,2,3,4-tetrahydroisoquinoline-3-carboxylate(Example 13-2b) and1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazole-4-carbonyl azide(Example 10-1a). Yield 84%, yellowish solid. MS 508 (MH⁺).

Example 13-2b (R)-methyl7-(tert-butyldimethylsilyloxy)-1,2,3,4-tetrahydroisoquinoline-3-carboxylate

Prepared as in Example 1c-JF from (R)-methyl7-hydroxy-1,2,3,4-tetrahydroisoquinoline-3-carboxylate hydrochloride(Example 13-2c) and tert-butylchlorodimethylsilane. Yield 79%, yellowoil (1.15 g, 79%). MS 322 (MH⁺).

Example 13-2c (R)-methyl7-hydroxy-1,2,3,4-tetrahydroisoquinoline-3-carboxylate hydrochloride

Prepared by refluxing(R)-7-hydroxy-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid in aconcentrated methanolic solution of HCl. Yield 100%, beige powder. MS208 (MH⁺—Cl⁻).

Example 13-32-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-8-hydroxy-10,10a-dihydroimidazo[1,5-b]isoquinoline-1,3(2H,5H)-dione

Prepared as in Example 12-1 from8-(tert-butyldimethylsilyloxy)-2-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-10,10a-dihydroimidazo[1,5-b]isoquinoline-1,3(2H,5H)-dione (Example 13-3a). Yield 96%, a white solid. MS 394 (MH⁺). Thetitle compound was shown to inhibit hT2R08 bitter receptor and had anIC₅₀ of 0.03 μM

Example 13-3a8-(tert-butyldimethylsilyloxy)-2-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-10,10a-dihydroimidazo[1,5-b]isoquinoline-1,3(2H,5H)-dione

Prepared as in Example 12-1a from methyl6-(tert-butyldimethylsilyloxy)-1,2,3,4-tetrahydroisoquinoline-3-carboxylate(Example 13-3b) and1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazole-4-carbonyl azide(Example 10-1a). Yield 90%, yellowish solid. MS 508 (MH⁺).

Example 13-3b methyl6-(tert-butyldimethylsilyloxy)-1,2,3,4-tetrahydroisoquinoline-3-carboxylate

Prepared as in Example 12-1c from methyl6-hydroxy-1,2,3,4-tetrahydroisoquinoline-3-carboxylate hydrochloride(Example 13-3c) and tert-butylchlorodimethylsilane. Yield 83%,yellowish. MS 322 (MH⁺).

Example 13-3c methyl6-hydroxy-1,2,3,4-tetrahydroisoquinoline-3-carboxylate hydrochloride

Prepared by refluxing6-hydroxy-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid in aconcentrated methanolic solution of HCl. Yield 100%, beige powder. MS208 (MH⁺—Cl⁻).

Example 14 hT2R8 Antagonists Reduce Bitter Taste of Instant Coffee

Taste tests were performed with hT2R8 antagonists in instant coffeeusing a 2-alternative forced choice method with a taste panel of 15-20panelists. Coffee samples with the antagonists were given to the tastepanelists together with the same sample without antagonists, thepanelists were asked to identify the bitterer sample within the pair. Asshown in Table 9, the panelists consistently identified the coffeefraction samples without antagonists as being bitterer than the oneswith antagonists, indicating that the antagonists reduced the bittertaste of instant coffee.

TABLE 9 Taste test results with Instant Coffee and T2R8 antagonistsSelected as bitterer Concentration Without With T2R8 Antagonist (μM)antagonists antagonists P value Example 10-10 5 14 2 0.004 Example 12-15 14 2 0.004 Example 12-34 5 13 3 0.021 Example 12-17 5 17 3 0.003Example 12-2 5 14 2 0.004

Other exemplary compounds provided by the present invention and/orsuitable to be used for methods of the present invention includecompounds of the following formulae.

In a first aspect, a compound of structural Formula (I) is provided:

or a salt, hydrate, solvate or N-oxide thereof wherein:

Ar¹ a five or six membered aryl, heteroaryl or cycloalkyl ring;

m is 0, 1, 2 or 3;

R¹ is SO₂; C═O; C═S; or C═NOR⁴;

X is selected from the group consisting of hydrogen, halogen, alkyl,substituted alkyl, aryl, substituted aryl, arylalkyl, substitutedarylalkyl, acyl, substituted acyl, heteroalkyl, substituted heteroalkyl,heteroaryl, substituted heteroaryl, heteroarylalkyl, substitutedheteroarylalkyl, CN, NO₂, OR⁶, S(O)_(b)R⁶, NR⁶R⁷, CONR⁶R⁷, CO₂R⁶,NR⁶CO₂R⁷, NR⁶CONR⁷R⁸, NR⁶CSNR⁷R⁸, NR⁶C(═NH)NR⁷R⁸, SO₂NR⁵R⁶, NR⁵SO₂R⁶,NR⁵SO₂NR⁶R⁷, B(OR⁵)(OR⁶), P(O)(OR⁵)(OR⁶), and P(O)(R⁵)(OR⁶);

each R¹′ is independently selected from the group consisting ofhydrogen, halogen, alkyl, substituted alkyl, aryl, substituted aryl,arylalkyl, substituted arylalkyl, acyl, substituted acyl, heteroalkyl,substituted heteroalkyl, heteroaryl, substituted heteroaryl,heteroarylalkyl, substituted heteroarylalkyl, CN, NO₂, OR⁶, S(O)_(b)R⁶,NR⁶R⁷, CONR⁶R⁷, CO₂R⁶, NR⁶CO₂R⁷, NR⁶CONR⁷R⁸, NR⁶CSNR⁷R⁸, NR⁶C(═NH)NR⁷R⁸,SO₂NR⁵R⁶, NR⁵SO₂R⁶, NR⁵SO₂NR⁶R⁷, B(OR⁵)(OR⁶), P(O)(OR⁵)(OR⁶), andP(O)(R⁵)(OR⁶);

or alternatively, X and/or at least one R¹′ together with the atoms towhich they are bonded form an aryl, substituted aryl, heteroaryl,substituted heteroaryl, cycloalkyl, substituted cycloalkyl,cycloheteroalkyl or substituted cycloheteroalkyl ring where the ring isoptionally fused to another aryl, substituted aryl, heteroaryl,substituted heteroaryl, cycloalkyl, substituted cycloalkyl,cycloheteroalkyl or substituted cycloheteroalkyl ring;

R⁴-R⁸ are independently selected from the group consisting of hydrogen,alkyl, substituted alkyl, aryl, substituted aryl, arylalkyl, substitutedarylalkyl, heteroalkyl, substituted heteroalkyl, heteroaryl, substitutedheteroaryl, heteroarylalkyl and substituted heteroarylalkyl oralternatively, R⁵ and R⁶, R⁶ and R⁷, R⁷ and R⁸, together with the atomsto which they are bonded form a cycloheteroalkyl or substitutedcycloheteroalkyl ring;

A and B are independently selected from the group consisting ofhydrogen, alkyl, substituted alkyl, aryl, substituted aryl, arylalkyl,substituted arylalkyl, acyl, substituted acyl, heteroalkyl, substitutedheteroalkyl, heteroaryl, substituted heteroaryl, heteroarylalkyl,substituted heteroarylalkyl; and

-   -   b is 0, 1, or 2;

In a second aspect the invention provides compounds of structuralFormula (II) shown below:

or a salt, hydrate, solvate or N-oxide thereof wherein:

Ar¹, Ar² and Ar³ are independently a five or six membered aryl,heteroaryl, or cycloalkyl ring;

m is 0, 1, 2 or 3;

n and p are independently 0, 1, 2, 3 or 4;

r and t are independently 0, 1 or 2;

Y and Z are independently selected from the group consisting of CR⁶R⁷,C═O, C═S, C═NOR⁶, O, NR⁶, and S(O)_(b);

R¹ is selected from the group consisting of SO₂, C═O, C═S, and C═NOR⁴;

X may be selected from the group consisting of hydrogen, alkyl,substituted alkyl, aryl, substituted aryl, arylalkyl, substitutedarylalkyl, acyl, substituted acyl, heteroalkyl, substituted heteroalkyl,heteroaryl, substituted heteroaryl, heteroarylalkyl, substitutedheteroarylalkyl, CN, NO₂, —OR⁶, S(O)_(b)R⁶, NR⁶R⁷, CONR⁶R⁷, CO₂R⁶,NR⁶CO₂R⁷, NR⁶CONR⁷R⁸, NR⁶CSNR⁷R⁸, NR⁶C(═NH)NR⁷R⁸, SO₂NR⁵R⁶, NR⁵SO₂R⁶,NR⁵SO₂NR⁶R⁷, B(OR⁵)(OR⁶), P(O)(OR⁵)(OR⁶), and P(O)(R⁵)(OR⁶);

X is preferably selected from the group consisting of hydrogen,heteroalkyl, substituted heteroalkyl, heteroaryl, substitutedheteroaryl, heteroarylalkyl, substituted heteroarylalkyl, CN,S(O)_(b)R⁶, CONR⁶R⁷, —CO₂R⁶, SO₂NR⁵R⁶, NR⁵SO₂R⁶, NR⁵SO₂NR⁶R⁷,B(OR⁵)(OR⁶), P(O)(OR⁵)(OR⁶), and P(O)(R⁵)(OR⁶).

each R¹′ is independently selected from the group consisting ofhydrogen, halogen, alkyl, substituted alkyl, aryl, substituted aryl,arylalkyl, substituted arylalkyl, acyl, substituted acyl, heteroalkyl,substituted heteroalkyl, heteroaryl, substituted heteroaryl,heteroarylalkyl, substituted heteroarylalkyl, CN, NO₂, OR⁶, S(O)_(b)R⁶,NR⁶R⁷, CONR⁶R⁷, CO₂R⁶, NR⁶CO₂R⁷, NR⁶CONR⁷R⁸, NR⁶CSNR⁷R⁸, NR⁶C(═NH)NR⁷R⁸,SO₂NR⁵R⁶, NR⁵SO₂R⁶, NR⁵SO₂NR⁶R⁷, B(OR⁵)(OR⁶), P(O)(OR⁵)(OR⁶), andP(O)(R⁵)(OR⁶);

each R²′ is independently selected from the group consisting ofhydrogen, halogen, alkyl, substituted alkyl, aryl, substituted aryl,arylalkyl, substituted arylalkyl, acyl, substituted acyl, heteroalkyl,substituted heteroalkyl, heteroaryl, substituted heteroaryl,heteroarylalkyl, substituted heteroarylalkyl, CN, NO₂, OR⁶, S(O)_(b)R⁶,NR⁶R⁷, CONR⁶R⁷, CO₂R⁶, NR⁶CO₂R⁷, NR⁶CONR⁷R⁸, NR⁶CSNR⁷R⁸, NR⁶C(═NH)NR⁷R⁸,SO₂NR⁵R⁶, NR⁵SO₂R⁶, NR⁵SO₂NR⁶R⁷, B(OR⁵)(OR⁶), and P(O)(OR⁵)(OR⁶);

each R³′ is independently selected from the group consisting ofhydrogen, halogen, alkyl, substituted alkyl, aryl, substituted aryl,arylalkyl, substituted arylalkyl, acyl, substituted acyl, heteroalkyl,substituted heteroalkyl, heteroaryl, substituted heteroaryl,heteroarylalkyl, substituted heteroarylalkyl, CN, NO₂, OR⁶, S(O)_(b)R⁶,NR⁶R⁷, CONR⁶R⁷, CO₂R⁶, NR⁶CO₂R⁷, NR⁶CONR⁷R⁸, NR⁶CSNR⁷R⁸, NR⁶C(═NH)NR⁷R⁸,SO₂NR⁵R⁶, NR⁵SO₂R⁶, NR⁵SO₂NR⁶R⁷, B(OR⁵)(OR⁶), P(O)(OR⁵)(OR⁶), andP(O)(R⁵)(OR⁶);

or alternatively, X and/or at least one of R¹′ together with the atomsto which they are bonded form an aryl, substituted aryl, heteroaryl,substituted heteroaryl, cycloalkyl, substituted cycloalkyl,cycloheteroalkyl or substituted cycloheteroalkyl ring where the ring isoptionally fused to another aryl, substituted aryl, heteroaryl,substituted heteroaryl, cycloalkyl, substituted cycloalkyl,cycloheteroalkyl or substituted cycloheteroalkyl ring;

R⁴-R⁸ are independently hydrogen, alkyl, substituted alkyl, aryl,substituted aryl, arylalkyl, substituted arylalkyl, heteroalkyl,substituted heteroalkyl, heteroaryl, substituted heteroaryl,heteroarylalkyl or substituted heteroarylalkyl or alternatively, R⁵ andR⁶, R⁶ and R⁷, R⁷ and R⁸, together with the atoms to which they arebonded form a cycloheteroalkyl or substituted cycloheteroalkyl ring;

b is 0, 1, or 2.

In another aspect the invention provides compounds having structuralFormula (III) shown below:

or a salt, hydrate, solvate or N-oxide thereof wherein:

Ar¹, Ar² and Ar³ are independently a five or six membered aryl,heteroaryl, or cycloalkyl ring, and Ar² and Ar³ may optionally beomitted;

m is 0, 1, 2 or 3;

n and p are independently 0, 1, 2, 3 or 4;

each R¹′ is independently selected from the group consisting ofhydrogen, halogen, alkyl, substituted alkyl, aryl, substituted aryl,arylalkyl, substituted arylalkyl, acyl, substituted acyl, heteroalkyl,substituted heteroalkyl, heteroaryl, substituted heteroaryl,heteroarylalkyl, substituted heteroarylalkyl, CN, NO₂, OR⁶, S(O)_(b)R⁶,NR⁶R⁷, CONR⁶R⁷, CO₂R⁶, NR⁶CO₂R⁷, NR⁶CONR⁷R⁸, NR⁶CSNR⁷R⁸, NR⁶C(═NH)NR⁷R⁸,SO₂NR⁵R⁶, NR⁵SO₂R⁶, NR⁵SO₂NR⁶R⁷, B(OR⁵)(OR⁶), P(O)(OR⁵)(OR⁶), andP(O)(R⁵)(OR⁶);

each R²′ is independently selected from the group consisting ofhydrogen, halogen, alkyl, substituted alkyl, aryl, substituted aryl,arylalkyl, substituted arylalkyl, acyl, substituted acyl, heteroalkyl,substituted heteroalkyl, heteroaryl, substituted heteroaryl,heteroarylalkyl, substituted heteroarylalkyl, CN, NO₂, OR⁶, S(O)_(b)R⁶,NR⁶R⁷, CONR⁶R⁷, CO₂R⁶, NR⁶CO₂R⁷, NR⁶CONR⁷R⁸, NR⁶CSNR⁷R⁸, NR⁶C(═NH)NR⁷R⁸,SO₂NR⁵R⁶, NR⁵SO₂R⁶, NR⁵SO₂NR⁶R⁷, B(OR⁵)(OR⁶), P(O)(OR⁵)(OR⁶), andP(O)(R⁵)(OR⁶);

each R³′ is independently selected from the group consisting ofhydrogen, halogen, alkyl, substituted alkyl, aryl, substituted aryl,arylalkyl, substituted arylalkyl, acyl, substituted acyl, heteroalkyl,substituted heteroalkyl, heteroaryl, substituted heteroaryl,heteroarylalkyl, substituted heteroarylalkyl, CN, NO₂, OR⁶, S(O)_(b)R⁶,NR⁶R⁷, CONR⁶R⁷, CO₂R⁶, NR⁶CO₂R⁷, NR⁶CONR⁷R⁸, NR⁶CSNR⁷R⁸, NR⁶C(═NH)NR⁷R⁸,SO₂NR⁵R⁶, NR⁵SO₂R⁶, NR⁵SO₂NR⁶R⁷, B(OR⁵)(OR⁶), P(O)(OR⁵)(OR⁶), andP(O)(R⁵)(OR⁶);

R⁵′R⁸ are independently hydrogen, alkyl, substituted alkyl, aryl,substituted aryl, arylalkyl, substituted arylalkyl, heteroalkyl,substituted heteroalkyl, heteroaryl, substituted heteroaryl,heteroarylalkyl or substituted heteroarylalkyl or alternatively, R⁵ andR⁶, R⁶ and R⁷, R⁷ and R⁸, together with the atoms to which they arebonded form a cycloheteroalkyl or substituted cycloheteroalkyl ring;

b is 0, 1, or 2.

In yet another aspect the invention provides a compound having thestructure below:

or a salt, hydrate, solvate or N-oxide thereof.

In yet another aspect the invention provides compounds having thestructure below:

or a salt, hydrate, solvate or N-oxide thereof.

In still other embodiments, the invention provides compounds having thestructure below:

or a salt, hydrate, solvate or N-oxide thereof.

In a related aspect, a compound of structural Formula (IV) is provided:

or a salt, hydrate, solvate or N-oxide thereof wherein:

Ar⁴ and Ar⁵ are independently a five or six membered aryl or heteroarylring;

W is selected from the group consisting of CR⁶R⁷, C═O, C═S; C═NOR⁶; O,NR⁶, S, SO, SO₂, and (CH₂)_(n);

n is 0, 1, 2, or 3;

G is selected from the group consisting of CR⁶R⁷, C═O, C═S, C═NOR⁶, andS(O)_(b);

R²⁰ is selected from the group consisting of hydrogen, arylalkenyl,heteroarylalkenyl, arylalkyl, heteroarylalkyl, aryl, heteroaryl, alkyl,alkoxy-alkyl, alkenyl, cycloalkyl, cycloalkenyl, and substitutedderivatives;

R²¹ is selected from the group consisting of an arylalkenyl,heteroarylalkenyl, arylalkyl, heteroarylalkyl, aryl, heteroaryl, alkyl,alkoxy-alkyl, alkenyl, cycloalkyl, cycloalkenyl, and substitutedderivatives;

R⁶ and R⁷ are independently selected from the group consisting ofhydrogen, alkyl, substituted alkyl, aryl, substituted aryl, arylalkyl,substituted arylalkyl, heteroalkyl, substituted heteroalkyl, heteroaryl,substituted heteroaryl, heteroarylalkyl or substituted heteroarylalkylor alternatively, R⁶ and R⁷, together with the atoms to which they arebonded form a cycloheteroalkyl or substituted cycloheteroalkyl ring; and

b is 0, 1, or 2.

In another related aspect a compound of structural Formula (V) isprovided:

or a salt, hydrate, solvate or N-oxide thereof wherein:

Ar⁴ and Ar⁵ are independently a five or six membered aryl or heteroarylring;

n is 0, 1, 2, or 3;

R²¹ is selected from the group consisting of an arylalkenyl,heteroarylalkenyl, arylalkyl, hereoarylalky, aryl, heteroaryl, alkyl,alkoxy-alkyl, alkenyl, cycloalkyl, cycloalkenyl, and substitutedderivatives;

R³⁵ is selected from the group consisting of hydrogen, alkyl, andsubstituted alkyl.

In still additional embodiments the invention a compound of structuralFormula (VI) is provided

or a salt, hydrate, solvate or N-oxide thereof wherein:

R³⁰ is selected from the group consisting of an arylalkenyl,heteroarylalkenyl, arylalkyl, hereoarylalky, aryl, heteroaryl, alkyl,alkoxy-alkyl, alkenyl, cycloalkyl, cycloalkenyl, and substitutedderivatives;

R³⁵ is selected from the group consisting of hydrogen, alkyl, andsubstituted alkyl.

In still additional embodiments the invention provides compounds havingthe structure below:

or a salt, hydrate, solvate or N-oxide thereof,wherein each R is independently Cl, MeO, CN, EtO, OH, Me, —SO₂Me, F, orH, andn is 0, 1, 2, 3 or 4.

In still other embodiments, the invention provides compounds having thestructure below:

or a salt, hydrate, solvate or N-oxide thereof,wherein each R is independently MeO or OH, andn is 0, 1, 2, 3 or 4.

In still other embodiments, the invention provides compounds having thestructure below:

or a salt, hydrate, solvate or N-oxide thereof,wherein R is H, Me, Et, OCOMe, CH₂OH, OMe, or Ph.

In still other embodiments, the invention provides compounds having thestructure below:

or a salt, hydrate, solvate or N-oxide thereof.

In still other embodiments, the invention provides compounds having thestructure below:

or a salt, hydrate, solvate or N-oxide thereof.

In still other embodiments, the invention provides compounds having thestructure below:

or a salt, hydrate, solvate or N-oxide thereof.

In still other embodiments, the invention provides compounds having thestructure below:

In one aspect, the invention relates to a compound of the formula:

-   -   or a salt, hydrate, solvate, N-oxide or prodrug thereof,    -   wherein Ar⁶ and Ar⁷ are, the same or different independently one        from the other, a five- or six-membered aryl group or a five- or        six-membered heteroaryl group;    -   Alk is an alkyl group, optionally interrupted by a heteroatom;    -   R₃₆ and R₃₇ are, the same or different independently one from        the other, H, alkyl, or, R₃₆ and R₃₇, together with the atoms to        which they are attached, form an optionally substituted five- or        six-membered heterocycle; and    -   R₃₈ is H, substituted or unsubstituted alkyl, substituted or        unsubstituted cycloalkyl alkyl, substituted or unsubstituted        heterocycloalkylalkyl, substituted or unsubstituted aryl,        substituted or unsubstituted arylamidoalkyl, substituted or        unsubstituted heteroarylamidoalkyl, substituted or unsubstituted        arylalkyl, substituted or unsubstituted arylalkoxy, substituted        or unsubstituted heteroaryl, substituted or unsubstituted        heteroarylalkyl, or haloalkyl.

In one aspect the compounds of the invention contain a five-memberedheterocycle. In one embodiment, the five-membered heterocycle is ahydantoin or a substituted or unsubstituted cyclic urea.

In one embodiment, the hydantoin is a hydantoin of the formula:

-   -   or a salt, hydrate, solvate, N-oxide or prodrug thereof,    -   wherein Ar⁶ and Ar⁷ are, the same or different independently one        from the other, a five- or six-membered aryl group or a five- or        six-membered heteroaryl group;    -   Alk is an alkyl group, optionally interrupted by a heteroatom;    -   R₃₈ is H, substituted or unsubstituted alkyl, substituted or        unsubstituted cycloalkyl alkyl, substituted or unsubstituted        heterocycloalkylalkyl, substituted or unsubstituted aryl,        substituted or unsubstituted arylamidoalkyl, substituted or        unsubstituted heteroarylamidoalkyl, substituted or unsubstituted        arylalkyl, substituted or unsubstituted arylalkoxy, substituted        or unsubstituted heteroaryl, substituted or unsubstituted        heteroarylalkyl, or haloalkyl; and    -   R₃₉ and R₄₀ are, the same or different independently one from        the other, H, substituted or unsubstituted alkyl, substituted or        unsubstituted cycloalkyl alkyl, substituted or unsubstituted        heterocycloalkylalkyl, substituted or unsubstituted aryl,        substituted or unsubstituted arylamidoalkyl, substituted or        unsubstituted aryl alkylamidoalkyl, substituted or unsubstituted        heteroarylamidoalkyl, substituted or unsubstituted        heteroarylalkylamidoalkyl, substituted or unsubstituted        arylalkyl, substituted or unsubstituted arylalkoxy, substituted        or unsubstituted heteroaryl, substituted or unsubstituted        heteroarylalkyl, haloalkyl, or R₃₉ and R₄₀, together with the        carbon atom to which they are attached, form a C═O group or a        substituted or unsubstituted alkenyl group.

In another aspect the compounds of the invention contain a five-memberedheterocycle which is a urazole. In one embodiment, the urazole is aurazole of the formula:

-   -   or a salt, hydrate, solvate, N-oxide or prodrug thereof,    -   wherein Ar⁶ and Ar⁷ are, the same or different independently one        from the other, a five- or six-membered aryl group or a five- or        six-membered heteroaryl group;    -   Alk is an alkyl group, optionally interrupted by a heteroatom;    -   R₃₈ is H, substituted or unsubstituted alkyl, substituted or        unsubstituted cycloalkyl alkyl, substituted or unsubstituted        heterocycloalkylalkyl, substituted or unsubstituted aryl,        substituted or unsubstituted arylamidoalkyl, substituted or        unsubstituted heteroarylamidoalkyl, substituted or unsubstituted        arylalkyl, substituted or unsubstituted arylalkoxy, substituted        or unsubstituted heteroaryl, substituted or unsubstituted        heteroarylalkyl, or haloalkyl; and    -   R₄₁ is H, substituted or unsubstituted alkyl, substituted or        unsubstituted cycloalkyl alkyl, substituted or unsubstituted        heterocycloalkylalkyl, substituted or unsubstituted aryl,        substituted or unsubstituted arylamidoalkyl, substituted or        unsubstituted arylalkylamidoalkyl, substituted or unsubstituted        heteroarylamidoalkyl, substituted or unsubstituted        heteroarylalkylamidoalkyl, substituted or unsubstituted        arylalkyl, substituted or unsubstituted arylalkoxy, substituted        or unsubstituted heteroaryl, substituted or unsubstituted        heteroarylalkyl, or haloalkyl.

In another aspect the compounds of the invention contain a six-memberedheterocycle. In one embodiment, the six-membered heterocycle is asix-membered heterocycle of the formula:

-   -   or a salt, hydrate, solvate, N-oxide or prodrug thereof,    -   wherein R₃₈ is H, substituted or unsubstituted alkyl,        substituted or unsubstituted cycloalkyl alkyl, substituted or        unsubstituted heterocycloalkylalkyl, substituted or        unsubstituted aryl, substituted or unsubstituted arylamidoalkyl,        substituted or unsubstituted heteroarylamidoalkyl, substituted        or unsubstituted arylalkyl, substituted or unsubstituted        arylalkoxy, substituted or unsubstituted heteroaryl, substituted        or unsubstituted heteroarylalkyl, or haloalkyl; and    -   R₄₂, R₄₃, R₄₄, R₄₅, and R₄₆ are, the same or different        independently one from the other, H, substituted or        unsubstituted alkyl, substituted or unsubstituted cycloalkyl        alkyl, substituted or unsubstituted arylalkyl, substituted or        unsubstituted arylalkoxy, substituted or unsubstituted        heteroaryl, substituted or unsubstituted heteroarylalkyl, or R₄₂        and R₄₃, or    -   R₄₅ and R₄₆, together with the carbon atoms to which each are        attached, form a C═O group.

In still another aspect, the invention relates to a compound of theformula:

-   -   or a salt, hydrate, solvate, N-oxide or prodrug thereof,    -   wherein Alk is an alkyl group, optionally interrupted by a        heteroatom;    -   M¹ is N or CR₄₉, wherein R₄₉ is H or substituted or        unsubstituted alkyl;    -   M² is N or CR₅₀, wherein R₅₀ is H or substituted or        unsubstituted alkyl;    -   R₃₆ and R₃₇ are, the same or different independently one from        the other, H, alkyl, or, R₃₆ and R₃₇, together with the atoms to        which they are attached, form an optionally substituted five- or        six-membered heterocycle; and    -   R₃₈ is H, substituted or unsubstituted alkyl, substituted or        unsubstituted cycloalkyl alkyl, substituted or unsubstituted        heterocycloalkylalkyl, substituted or unsubstituted aryl,        substituted or unsubstituted arylamidoalkyl, substituted or        unsubstituted heteroarylamidoalkyl, substituted or unsubstituted        arylalkyl, substituted or unsubstituted arylalkoxy, substituted        or unsubstituted heteroaryl, substituted or unsubstituted        heteroarylalkyl, or haloalkyl;    -   R₄₇ is H, substituted or unsubstituted alkyl, substituted or        unsubstituted alkoxy, substituted or unsubstituted aryl,        substituted or unsubstituted arylalkyl, or halo; and    -   R₄₈ is H, substituted or unsubstituted alkyl, substituted or        unsubstituted alkoxy, substituted or unsubstituted aryl,        substituted or unsubstituted arylalkyl, or halo.

In still another aspect, the invention relates to a compound of theformula:

-   -   or a salt, hydrate, solvate, N-oxide or prodrug thereof,    -   wherein Alk is an alkyl group, optionally interrupted by a        heteroatom;    -   T₁ is C═O and Q is CR₅₁R₅₂ or NR₅₁, wherein R₅₁ and R₅₂ are, the        same or different independently one from the other, H,        substituted or unsubstituted alkyl, substituted or unsubstituted        cycloalkyl alkyl, substituted or unsubstituted        heterocycloalkylalkyl, substituted or unsubstituted aryl,        substituted or unsubstituted arylamidoalkyl, substituted or        unsubstituted arylalkylamidoalkyl, substituted or unsubstituted        heteroarylamidoalkyl, substituted or unsubstituted        heteroarylalkylamidoalkyl, substituted or unsubstituted        arylalkyl, substituted or unsubstituted arylalkoxy, substituted        or unsubstituted heteroaryl, substituted or unsubstituted        heteroarylalkyl, haloalkyl, or R₅₁ and R₅₂, together with the        carbon atom to which they are attached, form a C═O group or a        substituted or unsubstituted alkenyl group;    -   M¹ is N or CR₄₉, wherein R₄₉ is H or substituted or        unsubstituted alkyl;    -   M² is N or CR₅₀, wherein R₅₀ is H or substituted or        unsubstituted alkyl;    -   R₃₈ is H, substituted or unsubstituted alkyl, substituted or        unsubstituted cycloalkyl alkyl, substituted or unsubstituted        heterocycloalkylalkyl, substituted or unsubstituted aryl,        substituted or unsubstituted arylamidoalkyl, substituted or        unsubstituted heteroarylamidoalkyl, substituted or unsubstituted        arylalkyl, substituted or unsubstituted arylalkoxy, substituted        or unsubstituted heteroaryl, substituted or unsubstituted        heteroarylalkyl, or haloalkyl;    -   R₄₇ is H, substituted or unsubstituted alkyl, substituted or        unsubstituted alkoxy, substituted or unsubstituted aryl,        substituted or unsubstituted arylalkyl, or halo; and    -   R₄₈ is H, substituted or unsubstituted alkyl, substituted or        unsubstituted alkoxy, substituted or unsubstituted aryl,        substituted or unsubstituted arylalkyl, or halo.

In still another aspect, the invention relates to a compound of theformula:

or a salt, hydrate, solvate, N-oxide or prodrug thereof.

In still another aspect, the invention relates to a compound of theformula:

or a salt, hydrate, solvate, N-oxide or prodrug thereof.

In still another aspect, the invention relates to a method of making acompound of the formula:

-   -   or a salt, hydrate, solvate, N-oxide or prodrug thereof,    -   wherein Alk is an alkyl group, optionally interrupted by a        heteroatom;    -   T₂ is C═S, C═O, or S(O)₂;    -   R₅₃ is substituted or unsubstituted alkyl, substituted or        unsubstituted cycloalkyl, substituted or unsubstituted        heteroaryl, substituted or unsubstituted aryl, or substituted or        unsubstituted arylalkyl;    -   M¹ is N or CR₅₄, wherein R₅₄ is H or substituted or        unsubstituted alkyl;    -   M² is N or CR₅₅, wherein R₅₅ is H or substituted or        unsubstituted alkyl;    -   R₅₆ is H, substituted or unsubstituted alkyl, substituted or        unsubstituted alkoxy, substituted or unsubstituted aryl,        substituted or unsubstituted arylalkyl, or halo; and    -   R₅₇ is H, substituted or unsubstituted alkyl, substituted or        unsubstituted alkoxy, substituted or unsubstituted aryl,        substituted or unsubstituted arylalkyl, or halo;    -   wherein the method comprises reacting a compound of the formula:

-   -   wherein R₅₆, R₅₇, and Alk are defined above and J is a leaving        group;    -   with a compound of the formula:

-   -   wherein M¹ and M² are defined above to give a compound of the        formula

-   -   having an NO₂ group;    -   reducing the NO₂ group to give a compound having an NH₂ group;        and    -   reacting the compound having an NH₂ group with a compound of the        formula

-   -   wherein J₂ is a leaving group and T₂ and R₅₃ are defined above.

In still another aspect, the invention relates to a method of making acompound of the formula:

-   -   or a salt, hydrate, solvate, N-oxide or prodrug thereof,    -   wherein Alk is an alkyl group, optionally interrupted by a        heteroatom;    -   R₅₁ and R₅₂ are, the same or different independently one from        the other, H, substituted or unsubstituted alkyl, substituted or        unsubstituted cycloalkyl alkyl, substituted or unsubstituted        heterocycloalkylalkyl, substituted or unsubstituted aryl,        substituted or unsubstituted arylamidoalkyl, substituted or        unsubstituted aryl alkylamidoalkyl, substituted or unsubstituted        heteroarylamidoalkyl, substituted or unsubstituted        heteroarylalkylamidoalkyl, substituted or unsubstituted        arylalkyl, substituted or unsubstituted arylalkoxy, substituted        or unsubstituted heteroaryl, substituted or unsubstituted        heteroarylalkyl, haloalkyl, or R₅₁ and R₅₂, together with the        carbon atom to which they are attached, form a substituted or        unsubstituted alkenyl group;    -   M¹ is N or CR₄₉, wherein R₄₉ is H or substituted or        unsubstituted alkyl;    -   M² is N or CR₅₀, wherein R₅₀ is H or substituted or        unsubstituted alkyl;    -   R₃₈ is H, substituted or unsubstituted alkyl, substituted or        unsubstituted cycloalkyl alkyl, substituted or unsubstituted        heterocycloalkylalkyl, substituted or unsubstituted aryl,        substituted or unsubstituted arylamidoalkyl, substituted or        unsubstituted heteroarylamidoalkyl, substituted or unsubstituted        arylalkyl, substituted or unsubstituted arylalkoxy, substituted        or unsubstituted heteroaryl, substituted or unsubstituted        heteroarylalkyl, or haloalkyl;    -   R₄₇ is H, substituted or unsubstituted alkyl, substituted or        unsubstituted alkoxy, substituted or unsubstituted aryl,        substituted or unsubstituted arylalkyl, or halo; and    -   R₄₈ is H, substituted or unsubstituted alkyl, substituted or        unsubstituted alkoxy, substituted or unsubstituted aryl,        substituted or unsubstituted arylalkyl, or halo;    -   wherein the method comprises heating a compound of the formula:

-   -   wherein R₄₇, R₄₈, Alk, M¹, and M² are defined above;    -   to convert the —CON₃ group to a —N═C═O group, and then reacting        with a compound of the formula:

-   -   wherein J₃ is a leaving group and R₃₈, R₅₁, and R₅₂ are defined        above.

In still another aspect, the invention relates to a method of making acompound of the formula:

-   -   or a salt, hydrate, solvate, N-oxide or prodrug thereof,    -   wherein Alk is an alkyl group, optionally interrupted by a        heteroatom;    -   R₅₂ is H, substituted or unsubstituted alkyl, substituted or        unsubstituted cycloalkyl alkyl, substituted or unsubstituted        heterocycloalkylalkyl, substituted or unsubstituted aryl,        substituted or unsubstituted arylamidoalkyl, substituted or        unsubstituted arylalkylamidoalkyl, substituted or unsubstituted        heteroarylamidoalkyl, substituted or unsubstituted        heteroarylalkylamidoalkyl, substituted or unsubstituted        arylalkyl, substituted or unsubstituted arylalkoxy, substituted        or unsubstituted heteroaryl, substituted or unsubstituted        heteroarylalkyl, haloalkyl;    -   M¹ is N or CR₄₉, wherein R₄₉ is H or substituted or        unsubstituted alkyl;    -   M² is N or CR₅₀, wherein R₅₀ is H or substituted or        unsubstituted alkyl;    -   R₃₈ is H, substituted or unsubstituted alkyl, substituted or        unsubstituted cycloalkyl alkyl, substituted or unsubstituted        heterocycloalkylalkyl, substituted or unsubstituted aryl,        substituted or unsubstituted arylamidoalkyl, substituted or        unsubstituted heteroarylamidoalkyl, substituted or unsubstituted        arylalkyl, substituted or unsubstituted arylalkoxy, substituted        or unsubstituted heteroaryl, substituted or unsubstituted        heteroarylalkyl, or haloalkyl;    -   R₄₇ is H, substituted or unsubstituted alkyl, substituted or        unsubstituted alkoxy, substituted or unsubstituted aryl,        substituted or unsubstituted arylalkyl, or halo; and    -   R₄₈ is H, substituted or unsubstituted alkyl, substituted or        unsubstituted alkoxy, substituted or unsubstituted aryl,        substituted or unsubstituted arylalkyl, or halo;    -   wherein the method comprises heating a compound of the formula:

-   -   wherein R₄₇, R₄₈, Alk, M¹, and M² are defined above;    -   to covert the —CON₃ group to a —N═C═O group, and then reacting        with a hydrazine of the formula:

-   -   wherein R₃₈ is defined above.

In still another aspect, the invention relates to a method of making acompound of the formula:

-   -   or a salt, hydrate, solvate or N-oxide thereof wherein:    -   Ar¹, Ar² and Ar³ are independently a five or six membered aryl,        heteroaryl, or cycloalkyl ring, and Ar² and Ar³ may optionally        be omitted;    -   m is 0, 1, 2 or 3;    -   n and p are independently 0, 1, 2, 3 or 4;    -   each R¹′ is independently selected from the group consisting of        hydrogen, halogen, alkyl, substituted alkyl, aryl, substituted        aryl, arylalkyl, substituted arylalkyl, acyl, substituted acyl,        heteroalkyl, substituted heteroalkyl, heteroaryl, substituted        heteroaryl, heteroarylalkyl, substituted heteroarylalkyl, CN,        NO₂, OR⁶, S(O)_(b)R⁶, NR⁶R⁷, CONR⁶R⁷, CO₂R⁶, NR⁶CO₂R⁷,        NR⁶CONR⁷R⁸, NR⁶CSNR⁷R⁸, NR⁶C(═NH)NR⁷R⁸, SO₂NR⁵R⁶, NR⁵SO₂R⁶,        NR⁵SO₂NR⁶R⁷, B(OR⁵)(OR⁶), P(O)(OR⁵)(OR⁶), and P(O)(R⁵)(OR⁶);    -   each R²′ is independently selected from the group consisting of        hydrogen, halogen, alkyl, substituted alkyl, aryl, substituted        aryl, arylalkyl, substituted arylalkyl, acyl, substituted acyl,        heteroalkyl, substituted heteroalkyl, heteroaryl, substituted        heteroaryl, heteroarylalkyl, substituted heteroarylalkyl, CN,        NO₂, OR⁶, S(O)_(b)R⁶, NR⁶R⁷, CONR⁶R⁷, CO₂R⁶, NR⁶CO₂R⁷,        NR⁶CONR⁷R⁸, NR⁶CSNR⁷R⁸, NR⁶C(═NH)NR⁷R⁸, SO₂NR⁵R⁶, NR⁵SO₂R⁶,        NR⁵SO₂NR⁶R⁷, B(OR⁵)(OR⁶), P(O)(OR⁵)(OR⁶), and P(O)(R⁵)(OR⁶);    -   each R³′ is independently selected from the group consisting of        hydrogen, halogen, alkyl, substituted alkyl, aryl, substituted        aryl, arylalkyl, substituted arylalkyl, acyl, substituted acyl,        heteroalkyl, substituted heteroalkyl, heteroaryl, substituted        heteroaryl, heteroarylalkyl, substituted heteroarylalkyl, CN,        NO₂, OR⁶, S(O)_(b)R⁶, NR⁶R⁷, CONR⁶R⁷, CO₂R⁶, NR⁶CO₂R⁷,        NR⁶CONR⁷R⁸, NR⁶CSNR⁷R⁸, NR⁶C(═NH)NR⁷R⁸, SO₂NR⁵R⁶, NR⁵SO₂R⁶,        NR⁵SO₂NR⁶R⁷, B(OR⁵)(OR⁶), P(O)(OR⁵)(OR⁶), and P(O)(R⁵)(OR⁶);    -   R⁵-R⁸ are independently hydrogen, alkyl, substituted alkyl,        aryl, substituted aryl, arylalkyl, substituted arylalkyl,        heteroalkyl, substituted heteroalkyl, heteroaryl, substituted        heteroaryl, heteroarylalkyl or substituted heteroarylalkyl or        alternatively, R⁶ and R⁷, R⁷ and R⁸, together with the atoms to        which they are bonded form a cycloheteroalkyl or substituted        cycloheteroalkyl ring;    -   b is 0, 1, or 2;    -   wherein the method comprises reacting a compound of the formula:

-   -   wherein J is a leaving group;    -   with a compound of the formula:

-   -   to give a product; and    -   reacting the product with a compound of the formula:

-   -   wherein J is a leaving group.

Sequences of Chimeric G Protein and hT2R Genes and Polypeptides

Protein Sequence of the Rhodopsin tag: (SEQ ID NO: 1)MNGTEGPNFYVPFSNKTGVVRSPFEAPQYYLAEPW Protein Sequence of G16gust44: (SEQID NO: 2) MARSLTWRCCPWCLTEDEKAAARVDQEINRILLEQKKQDRGELKLLLLGPGESGKSTFIKQMRIIHGAGYSEEERKGFRPLVYQNIFVSMRAMIEAMERLQIPFSRPESKHHASLVMSQDPYKVTTFEKRYAAAMQWLWRDAGIRACYERRREFHLLDSAVYYLSHLERITEEGYVPTAQDVLRSRMPTTGINEYCFSVQKTNLRIVDVGGQKSERKKWIHCFENVIALIYLASLSEYDQCLEENNQENRMKESLALFGTILELPWFKSTSVILFLNKTDILEEKIPTSHLATYFPSFQGPKQDAEAAKRFILDMYTRMYTGCVDGPEGSNLKKEDKEIYSHMTCATDTQNVKFVFDAVTDIIIKENLKDCGLF hT2R8 sequences: DNA- (SEQ ID NO: 3)ATGTTCAGTCCTGCAGATAACATCTTTATAATCCTAATAACTGGAGAATTCATACTAGGAATATTGGGGAATGGATACATTGCACTAGTCAACTGGATTGACTGGATTAAGAAGAAAAAGATTTCCACAGTTGACTACATCCTTACCAATTTAGTTATCGCCAGAATTTGTTTGATCAGTGTAATGGTTGTAAATGGCATTGTAATAGTACTGAACCCAGATGTTTATACAAAAAATAAACAACAGATAGTCATTTTTACCTTCTGGACATTTGCCAACTACTTAAATATGTGGATTACCACCTGCCTTAATGTCTTCTATTTTCTGAAGATAGCCAGTTCCTCTCATCCACTTTTTCTCTGGCTGAAGTGGAAAATTGATATGGTGGTGCACTGGATCCTGCTGGGATGCTTTGCCATTTCCTTGTTGGTCAGCCTTATAGCAGCAATAGTACTGAGTTGTGATTATAGGTTTCATGCAATTGCCAAACATAAAAGAAACATTACTGAAATGTTCCATGTGAGTAAAATACCATACTTTGAACCCTTaACTCTCTTTAACCTGTTTGCAATTGTCCCATTTATTGTGTCACTGATATCATTTTTCCTTTTAGTAAGATCTTTATGGAGACATACCAAGCAAATAAAACTCTATGCTACCGGCAGTAGAGACCCCAGCACAGAAGTTCATGTGAGAGCCATTAAAACTATGACTTCATTTATCTTCTTTTTTTTCCTATACTATATTTCTTCTATTTTGATGACCTTTAGCTATCTTATGACAAAATACAAGTTAGCTGTGGAGTTTGGAGAGATTGCAGCAATTCTCTACCCCTTGGGTCACTCACTTATTTTAATTGTTTTAAATAATAAACTGAGGCAGACATTTGTCAGAATGCTGACATGTAGAAAAATTGCCTGCATGATATGA Protein- (SEQ ID NO:4) MFSPADNIFIILITGEFILGILGNGYIALVNWIDWIKKKKISTVDYILTNLVIARICLISVMVVNGIVIVLNPDVYTKNKQQIVIFTFWTFANYLNMWITTCLNVFYFLKIASSSHPLFLWLKWKIDMVVHWILLGCFAISLLVSLIAAIVLSCDYRFHAIAKHKRNITEMFHVSKIPYFEPLTLFNLFAIVPFIVSLISFFLLVRSLWRHTKQIKLYATGSRDPSTEVHVRAIKTMTSFIFFFFLYYISSILMTFSYLMTKYKLAVEFGEIAAILYPLGHSLILIVLNN KLRQTFVRMLTCRKIACMIhT2R14 sequences: DNA- (SEQ ID NO: 5)ATGGGTGGTGTCATAAAGAGCATATTTACATTCGTTTTAATTGTGGAATTTATAATTGGAAATTTAGGAAATAGTTTCATAGCACTGGTGAACTGTATTGACTGGGTCAAGGGAAGAAAGATCTCTTCGGTTGATCGGATCCTCACTGCTTTGGCAATCTCTCGAATTAGCCTGGTTTGGTTAATATTCGGAAGCTGGTGTGTGTCTGTGTTTTTCCCAGCTTTATTTGCCACTGAAAAAATGTTCAGAATGCTTACTAATATCTGGACAGTGATCAATCATTTTAGTGTCTGGTTAGCTACAGGCCTCGGTACTTTTTATTTTCTCAAGATAGCCAATTTTTCTAACTCTATTTTTCTCTACCTAAAGTGGAGaGTTAAAAAGGTGGTTTTGGTGCTGCTTCTTGTGACTTCGGTCTTCTTGTTTTTAAATATTGCACTGATAAACATCCATATAAATGCCAGTATCAATGGATACAGAAGAAACAAGACTTGCAGTTCTGATTCAAGTAACTTTACACGATTTTCCAGTCTTATTGTATTAACCAGCACTGTGTTCATTTTCATACCCTTTACTTTGTCCCTGGCAATGTTTCTTCTCCTCATCTTCTCCATGTGGAAACATCGCAAGAAGATGCAGCACACTGTCAAAATATCCGGAGACGCCAGCACCAAAGCCCACAGAGGAGTTAAAAGTGTGATCACTTTCTTCCTACTCTATGCCATTTTCTCTCTGTCTTTTTTCATATCAGTTTGGACCTCTGAAAGGTTGGAGGAAAATCTAATTATTCTTTCCCAGGTGATGGGAATGGCTTATCCTTCATGTCACTCATGTGTTCTGATTCTTGGAAACAAGAAGCTGAGACAGGCCTCTCTGTCAGTGCTACTGTGGCTGAGGTACATGTTCAAAGATGGGGAGCCCTCAGGTCACAAAGAATTTAGAGAATCATCTTGA Protein- (SEQ ID NO: 6)MGGVIKSIFTFVLIVEFIIGNLGNSFIALVNCIDWVKGRKISSVDRILTALAISRISLVWLIFGSWCVSVFFPALFATEKMFRMLTNIWTVINHFSVWLATGLGTFYFLKIANFSNSIFLYLKWRVKKVVLVLLLVTSVFLFLNIALINIHINASINGYRRNKTCSSDSSNFTRFSSLIVLTSTVFIFIPFTLSLAMFLLLIFSMWKHRKKMQHTVKISGDASTKAHRGVKSVITFFLLYAIFSLSFFISVWTSERLEENLIILSQVMGMAYPSCHSCVLILGNKKLRQASLSVLLWLRYMFKDGEPSGHKEFRESS

While the foregoing detailed description has described severalembodiments of the present invention, it is to be understood that theabove description is illustrative only and not limiting of the disclosedinvention. The invention is to be limited only by the claims whichfollow.

1. A compound of the formula:

or a salt or N-oxide thereof, wherein Ar⁶ and Ar⁷ are the same ordifferent, independently one from the other, and each is an optionallysubstituted five-membered heteroaryl group; Alk is an alkyl group; R₅₈is H, alkyl, substituted alkyl, aryl, substituted aryl, arylalkyl,substituted arylalkyl, acyl, substituted acyl, heteroalkyl, substitutedheteroalkyl, heteroaryl, substituted heteroaryl, heteroarylalkyl, orsubstituted heteroarylalkyl; R₅₉ and R₆₀ are the same or differentindependently one from the other, H, alkyl, substituted alkyl, aryl,substituted aryl, arylalkyl, substituted arylalkyl, acyl, substitutedacyl, heteroalkyl, substituted heteroalkyl, heteroaryl, substitutedheteroaryl, heteroarylalkyl or substituted heteroarylalkyl, with theproviso that when one of R₅₉ or R₆₀ is H, then the other of R₅₉ or R₆₀is not H; or R₅₉ and R₆₀, together with the atom to which they areattached form a cycloalkyl, a substituted cycloalkyl, a cycloheteroalkylor substituted cycloheteroalkyl ring; or R₅₈ and R₆₀, together with theatoms to which they are attached, form a five- or six-memberedcycloheteroalkyl or substituted cycloheteroalkyl ring.
 2. A compound ofthe formula:

a salt or N-oxide thereof, wherein: R₆₁ is H, alkyl, substituted alkyl,aryl, substituted aryl, arylalkyl, substituted arylalkyl, acyl,substituted acyl, heteroalkyl, substituted heteroalkyl, heteroaryl,substituted heteroaryl, heteroarylalkyl or substituted heteroarylalkyl;R₆₂ and R₆₃ are the same or different independently one from the other,H, alkyl, substituted alkyl, aryl, substituted aryl, arylalkyl,substituted arylalkyl, acyl, substituted acyl, heteroalkyl, substitutedheteroalkyl, heteroaryl, substituted heteroaryl, heteroarylalkyl orsubstituted heteroarylalkyl, with the proviso that when one of R₆₂ orR₆₃ is H, then the other of R₆₂ or R₆₃ is not H; or R₆₂ and R₆₃,together with the atoms to which they are attached form a cycloalkyl, asubstituted cycloalkyl, a cycloheteroalkyl or substitutedcycloheteroalkyl ring; or R₆₁ and R₆₂, together with the atoms to whichthey are attached, form a five- or six-membered cycloheteroalkyl orsubstituted cycloheteroalkyl ring.
 3. A compound of the formula:

a salt or N-oxide thereof, wherein: R₆₄ and R₆₅ are the same ordifferent independently one from the other, H, alkyl, substituted alkyl,aryl, substituted aryl, arylalkyl, substituted arylalkyl, acyl,substituted acyl, heteroalkyl, substituted heteroalkyl, heteroaryl,substituted heteroaryl, heteroarylalkyl or substituted heteroarylalkyl,with the proviso that when one of R₆₄ or R₆₅ is H, then the other of R₆₄or R₆₅ is not H; or R₆₄ and R₆₅, together with the atoms to which theyare attached, form a cycloalkyl, a substituted cycloalkyl, acycloheteroalkyl or substituted cycloheteroalkyl ring; each R₆₆ isindependently hydrogen, alkyl, substituted alkyl, aryl, substitutedaryl, arylalkyl, substituted arylalkyl, acyl, substituted acyl,heteroalkyl, substituted heteroalkyl, heteroaryl, substitutedheteroaryl, heteroarylalkyl, substituted heteroarylalkyl, F, Cl, —CN,—NO₂, —OR₆₇, —S(O)_(i)R₆₇, —NR₆₇R₆₈, —CONR₆₇R₆₈, —CO₂R₆₇, —NR₆₇CO₂R₆₈,—NR₆₇CONR₆₈R₆₉, —NR₆₇CSNR₆₈R₆₉ or —NR₆₇C(═NH)NR₆₈R₆₉, —SO₂NR₆₇R₆₈,—NR₆₇SO₂R₆₈, —NR₆₇SO₂NR₆₈R₆₉, —B(OR₆₇)(OR₆₈), —P(O)(OR₆₇)(OR₆₈), or—P(O)(R₆₇)(OR₆₈), wherein R₆₇, R₆₈, and R₆₉ are independently hydrogen,alkyl, substituted alkyl, aryl, substituted aryl, arylalkyl, substitutedarylalkyl, heteroalkyl, substituted heteroalkyl, heteroaryl, substitutedheteroaryl, heteroarylalkyl or substituted heteroarylalkyl oralternatively, R₆₇ and R₆₈ or R₆₈ and R₆₉, together with the atoms towhich they are attached form a cycloheteroalkyl or substitutedcycloheteroalkyl ring; Ar¹⁰ is an aryl or heteroaryl ring; g is 0, 1, 2,3 or 4; h is 0, 1, 2, 3 or 4; and i is 0, 1 or
 2. 4. A compound of theformula:

a salt or N-oxide thereof, wherein: each R₇₀ is independently hydrogen,alkyl, substituted alkyl, aryl, substituted aryl, arylalkyl, substitutedarylalkyl, acyl, substituted acyl, heteroalkyl, substituted heteroalkyl,heteroaryl, substituted heteroaryl, heteroarylalkyl, substitutedheteroarylalkyl, F, Cl, —CN, —NO₂, —OR₆₇, —S(O)_(i)R₆₇, —NR₆₇R₆₈,—CONR₆₇R₆₈, —CO₂R₆₇, —NR₆₇CO₂R₆₈, —NR₆₇CONR₆₈R₆₉, —NR₆₇CSNR₆₈R₆₉ or—NR₆₇C(═NH)NR₆₈R₆₉, —SO₂NR₆₇R₆₈, —NR₆₇SO₂R₆₈, —NR₆₇SO₂NR₆₈R₆₉,—B(OR₆₇)(OR₆₈), —P(O)(OR₆₇)(OR₆₈), or —P(O)(R₆₇)(OR₆₈), wherein R₆₇,R₆₈, and R₆₉ are independently hydrogen, alkyl, substituted alkyl, aryl,substituted aryl, arylalkyl, substituted arylalkyl, heteroalkyl,substituted heteroalkyl, heteroaryl, substituted heteroaryl,heteroarylalkyl or substituted heteroarylalkyl or alternatively, R₆₇ andR₆₈ or R₆₈ and R₆₉, together with the atoms to which they are attachedform a cycloheteroalkyl or substituted cycloheteroalkyl ring; j is 0, 1or 2; k is 0, 1 or 2; and l is 0, 1 or 2; with the proviso that j and kare not simultaneously both
 0. 5. The compound of claim 1 having theformula:

or a salt or N-oxide thereof.
 6. The compound of claim 1 having theformula:

or a salt or N-oxide thereof.
 7. A composition comprising one or morecompounds of one of claims 1-6 or a salt or N-oxide thereof and one ormore pharmaceutically acceptable carriers.
 8. The composition of claim7, which is a food, beverage or medicament for human consumption.
 9. Acoffee or coffee flavored food or beverage or medicament compositionthat comprises at least one compound of one of claims 1-6 or a salt orN-oxide thereof.
 10. The composition of claim 9, wherein at least onecompound is an hT2R8 antagonist.
 11. The composition of claim 9, whichis an instant coffee, ground coffee, or brewed coffee.
 12. Thecomposition of claim 9, which is an instant coffee.
 13. A food,beverage, or medicament composition having a bitter taste wherein saidbitter taste is alleviated or eliminated by the addition of an effectiveamount of a compound of one of claims 1-6 or a salt or N-oxide thereof.14. The composition of claim 13, wherein at least one compound is anhT2R8 antagonist.
 15. The composition of claim 13, wherein said beverageis an instant coffee beverage, a ground coffee beverage, or a brewedcoffee beverage.
 16. The composition of claim 13, wherein said beverageis an instant coffee beverage.
 17. An ingestible composition comprisingone or more compounds of claim 1 or a salt or N-oxide thereof.
 18. Thecomposition of claim 17, wherein said composition is a food or beverageproduct.
 19. The composition of claim 18, wherein the beverage productis a ready-to-drink coffee beverage, a soluble and dry coffee beverage,a coffee beverage mix, or a coffee beverage concentrate.
 20. Thecomposition of claim 18, wherein the beverage product is a dairy basedcreamer, a non-dairy based creamer or a whitener for coffee beverages.21. The composition of claim 17, wherein the composition is anon-comestible product.
 22. The composition of claim 21, wherein thenon-comestible product is a supplement, nutraceutical, functional foodproduct, pharmaceutical, over the counter medication, oral care productor a cosmetic product.
 23. The composition of claim 22, wherein the oralcare product is a dentifrice, mouthwash or chewing gum.